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The article presents a study on acaricide resistance and synergism between amitraz and permethrin against susceptible and resistant strains of Boophilus microplus (Acari: Ixodidae). It is posed that the control of the southern cattle tick, Boophilus microplus (Canestrini) in Mexico and some other countries depends on chemical acaricides. Relative to this, it is forwarded that Boophilus microplus has developed resistance to all major types of acaricides in recent years. In this study, the authors employed larval bioassay techniques to identify levels of resistance to amitraz and permethrin and then assessed synergism between these two acaricides in one susceptible laboratory tick strain and four resistant strains from Brazil and Mexico.

Abstract: The San Roman strain of the southern cattle tick, Boophilus microplus, collected from Mexico was previously reported to have a high level of resistance to the organophosphate acaricide coumaphos. An oxidative detoxification mechanism was suspected to contribute to coumaphos resistance in this tick strain, as coumaphos bioassay with piperonyl butoxide (PBO) on larvae of this resistant strain resulted in enhanced coumaphos toxicity, while coumaphos assays with PBO resulted in reduced toxicity of coumaphos in a susceptible reference strain. In this study, we further analyzed the mechanism of oxidative metabolic detoxification with synergist bioassays of coroxon, the toxic metabolite of coumaphos, and the mechanism of target-site insensitivity with acetylcholinesterase (AChE) inhibition kinetics assays. Bioassays of coroxon with PBO resulted in synergism of coroxon toxicity in both the San Roman and the susceptible reference strains. The synergism ratio of PBO on coroxon in the resistant strain was 4.5 times that of the susceptible strain. The results suggested that the cytP450-based metabolic detoxification existed in both resistant and susceptible strains, but its activity was significantly enhanced in the resistant strain. Comparisons of AChE activity and inhibition kinetics by coroxon in both susceptible and resistant strains revealed that the resistant San Roman strain had an insensitive AChE, with a reduced phosphorylation rate, resulting in a reduced bimolecular reaction constant. These data indicate a mechanism of coumaphos resistance in the San Roman strain that involves both insensitive AChE and enhanced cytP450-based metabolic detoxification. [Copyright &y& Elsevier]

We review recent progress for control of Rhipicephalus (Boophilus) microplus and R. (B.) annulatus. Outbreak infestations in the U.S. have reached alarming levels, due to increased populations of deer and other ungulates serving as alternative hosts. GIS mapping of infestations and deer habitat aids in utilizing methods and equipment designed for acaricide treatment of wild ungulates. Investigations include deer immunology, tick molecular genetics, acaricide resistance, and host-parasite interactions with deer or cattle, ticks, and pathogens. Acaricide resistance is widespread in Mexico and U.S. dipping vats depend on organophosphate (OP) inhibition of acetylcholinesterase (AChE). Three cDNAs putatively encoding acetylcholinesterases (AChE, E.C. 3.1.1.7) in R. (B.) microplus were expressed in the baculovirus system and exhibited Km values for acetylthiocholine of approx. 5, 50, or 90 µM, for rBmAChE1, rBmAChE2, and rBmAChE3, respectively. The rBmAChEs exhibited substrate preference for acetylthiocholine over butyrylthiocholine, and inhibition by eserine, paraoxon, and the AChE-specific inhibitor, BW284C51, confirming biochemical identification as AChEs. Expression of specific mutations from OP-resistant strains exhibited decreased sensitivity of rBmAChE1 and rBmAChE3 to OP inhibition. Each of the BmAChEs was expressed in synganglion as indicated by qRT-PCR. Multiple transcripts from individual ticks for each of the BmAChEs suggested alternative splicing or gene duplication. Quantitative real time PCR with genomic DNA supported the gene duplication hypothesis. Long dsRNA specific for BmAChE1, BmAChE2, and BmAChE3 was introduced by microinjection of unfed adult females and subsequent gene silencing was monitored by qRT-PCR and phenotypic effects. Specific physiological roles for BmAChE1, BmAChE2, and BmAChE3 remain to be elucidated. [ABSTRACT FROM AUTHOR]

Ticks and the diseases they transmit cause great economic losses to livestock in tropical countries. Non-chemical control alternatives include the use of resistant cattle breeds, biological control and vaccines. However, the most widely used method is the application of different chemical classes of acaricides and macrocyclic lactones. Populations of the cattle tick, Rhipicephalus (Boophilus) microplus, resistant to organophosphates (OP), synthetic pyrethroids (SP), amitraz and fipronil have been reported in Mexico. Macrocyclic lactones are the most sold antiparasitic drug in the Mexican veterinary market. Ivermectin-resistant populations of R. (B.) microplus have been reported in Brazil, Uruguay and especially in Mexico (Veracruz and Yucatan). Although ivermectin resistance levels in R. (B.) microplus from Mexico were generally low in most cases, some field populations of R. (B.) microplus exhibited high levels of ivermectin resistance. The CHPAT population showed a resistance ratio of 10.23 and 79.6 at lethal concentration of 50% and 99%, respectively. Many field populations of R. (B.) microplus are resistant to multiple classes of antiparasitic drugs, including organophosphates (chlorpyrifos, coumaphos and diazinon), pyrethroids (flumethrin, deltamethrin and cypermethrin), amitraz and ivermectin. This paper reports the current status of the resistance of R. (B.) microplus to acaricides, especially ivermectin, in Mexican cattle.

Boophilus microplus, collected from Nuevo Leon, Mexico, were found to be highly resistant to diazinon but not highly resistant to coumaphos, suggesting that different mechanisms of resistance were present in these ticks than other Mexican organophosphate (OP)-resistant ticks reported previously. When exposed to coumaphos and piperonyl butoxide or triphenylphosphate, the LCso estimate was reduced by 3.5- and 6.3-fold, respectively, suggesting that mono-oxygenases and/or esterases were involved in resistance to coumaphos. Additionally, it was determined that this strain had an Acetylycholinesterase (AChe) that was insensitive to the active form of coumaphos, coroxon, taking at least 24 min longer to reach 50% reduction in AChE activity compared with the susceptible strain. When exposed to diazinon, none of the synergists tested significantly lowered the LC50. However, it was determined that it took six times longer to reach 60% inhibition of AChE in the resistant strain compared with the susceptible strain when exposed to the active form of diazinon, diazoxon. Insensitive AChE seems to be very common in OP-resistant B. microplus. The potential benefits for the development of a field-portable AChE inhibition assay kit are discussed.

Susceptibility to carbaryl in six Mexican strains of the southern cattle tick, Boophilus microplus (Canestrini), was evaluated with the Food and Agricultural Organization larval packet test. Tick strains from the cattle fever tick quarantine zone in Texas were more susceptible to carbaryl than to coumaphos or diazinon. Compared with the susceptible reference (Gonzalez) strain, Mexican tick strains demonstrated 10.9-59.5-fold resistance to carbaryl. Significant cross-resistance was found between carbaryl and the organophosphate acaricides coumaphos and diazinon. Bioassay results with synergists suggested that metabolic detoxification mechanisms did not play a major role in carbaryl resistance. Resistance to carbaryl was likely conferred by insensitive acetylcholinesterase. The implications of carbaryl resistance in tick eradication and control also are discussed.

A field survey was conducted in 2001 to evaluate resistance to pyrethroid and organophosphate (OP) insecticides on horn flies, Hematobia irritans irritans (L.), from seven ranches in the state of Tamaulipas, Mexico, and from three locations in central Texas. Filter papers impregnated with either technical permethrin or diazinon were used to measure the levels of resistance to pyrethroids and OPs. A multiplex polymerase chain reaction (PCR) assay was used on individual horn flies from these field populations to detect the presence of the kdr and super-kdr alleles associated with pyrethroid resistance, and a mutated alphaE7 esterase allele associated with OP resistance. Relative to a susceptible laboratory (Kerrville) strain, horn flies from Mexico exhibited 5.1- to 28.3-fold resistance to permethrin at the LC50, and 23.8- to 136-fold resistance at the LC90. Horn flies from Texas ranches exhibited only two- to five-fold resistance. All field populations of the horn fly were highly susceptible to diazinon, and no mutant alphaE7 esterase alleles were detected. The super-kdr allele was found only in a single fly from a ranch in Mexico. Results of PCR assays showed that the kdr allele was present at various frequencies in field populations of horn flies. A gender-related bias in distribution of kdr genotypes was found in horn flies from Mexico, but not in horn flies from Texas. The overall kdr allelic frequencies in horn flies from Mexico were 23.2-37.8% higher in females than in males. Regression analysis revealed a significant correlation between kdr allelic frequencies and the levels of knockdown resistance to permethrin among the horn fly populations studied. The results validate the role of the PCR-based molecular assay as a diagnostic tool in monitoring resistance to pyrethroids and also provide useful information on population genetics of horn fly resistance to pyrethroids and OPs.

The levels of resistance to two organophosphate acaricides, coumaphos and diazinon, in several Mexican strains of Boophilus microplus (Canestrini) were evaluated using the FAO larval packet test. Regression analysis of LC50 data revealed a significant cross-resistance pattern between those two acaricides. Metabolic mechanisms of resistance were investigated with synergist bioassays. Piperonyl butoxide (PBO) reduced coumaphos toxicity in susceptible strains, but synergized coumaphos toxicity in resistant strains. There was a significant correlation between PBO synergism ratios and the coumaphos resistance ratios. The results suggest that an enhanced cytochrome P450 monooxygenase (cytP450)-mediated detoxification mechanism may exist in the resistant strains, in addition to the cytP450-mediated metabolic pathway that activates coumaphos. PBO failed to synergize diazinon toxicity in resistant strains, suggesting the cytP450 involved in detoxification were specific. Triphenylphosphate (TPP) synergized toxicity of both acaricides in both susceptible and resistant strains, and there was no correlation between TPP synergism ratios and the LC50 estimates for either acaricide. Esterases may not play a major role in resistance to coumaphos and diazinon in those strains. Bioassays with diethyl maleate (DEM) revealed a significant correlation between DEM synergism ratios and LC50 estimates for diazinon, suggesting a possible role for glutathione S-transferases in diazinon detoxification. Resistance to coumaphos in the Mexican strains of B. microplus was likely to be conferred by both a cytP450-mediated detoxification mechanism described here and the mechanism of insensitive acetylcholinesterases reported elsewhere. The results of this study also underscore the potential risk of coumaphos resistance in B. microplus from Mexico to the U.S. cattle fever tick eradication program.

Polymerase chain reaction (PCR) diagnostic assays were used to identify possible resistance-associated roles of two amino acid substitutions found in pyrethroid resistance-associated genes of Boophilus microplus (Canestrini). Individual larvae from the San Felipe target site resistant strain and the Coatzacoalcos (Cz) metabolic resistant strain were separated into resistant and susceptible groups by larval packet bioassays and analyzed by PCR. A Phe --> Ile amino acid mutation in the sodium channel gene S6 transmembrane segment of domain III was found to have a close association with survival of acaricide treatments containing as high as 30% permethrin. As the permethrin dose was increased, an increase was seen in the proportion of surviving larvae that possessed two mutated sodium channel alleles. An Asp --> Asn amino acid substitution, originally found in high allele frequency in alleles of the CzEst9 esterase of the Cz strain, appeared to provide some resistance to permethrin. However, the presence of the mutation did not associate with resistance in the dose-response fashion seen with the sodium channel amino acid mutation. Resistance provided by CzEst9 might be more dependent on concentration of CzEst9 more so than the presence of a mutated allele.

We examined pyrethroid resistant Mexican strains of Boophilus microplus using biochemical and molecular tests to determine the mechanisms conferring resistance. Permethrin hydrolysis assays and esterase activity gels indicated enhanced esterase-mediated metabolic detoxification in the Cz strain, while one other pyrethroid resistant strain, SF, and two pyrethroid susceptible strains had lower levels of permethrin hydrolysis. Results from assays using a PCR-based test to detect a pyrethroid target site resistance-associated mutation in the tick sodium channel gene found only low levels of mutations in the Cz strain, while the SF strain had a high level of the mutated sodium channel alleles. A specific esterase, designated CzEst9, believed to be responsible for the esterase-mediated pyrethroid resistance in the Cz strain was purified, and the gene encoding CzEst9 cloned.