In vitro Detection of Acaricide Resistance in Boophilus microplus (Acari: Ixodidae)

The cattle tick Boophilus microplus causes significant economic losses in cattle in the tropical areas of Central and South America. Acaricides are widely used to control tick infestations, with the consequent selection of tolerant and resistant subpopulations. Detoxifying enzymes, and esterases in particular, have been associated with organophosphate resistance in Mexican isolates of B. microplus ticks. Here, we present results of the biochemical detection of esterases and oxidases in 20 isolates of larvae and adult ticks and the effect of esterase and oxidase inhibitors on larvae and adult stages of the tick. Our results indicate the presence of significant differences both in vivo and in vitro between esterase and oxidase activities of OP-susceptible and OP-resistant isolates, even when samples were collected in the same area. The importance of such differences in regionalized integrated pest management and in the US Tick Eradication Program is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cloning and partial characterization of a Boophilus microplus (Acari: Ixodidae) calreticulin

We report the cloning, sequence characterization and expression analysis of a calreticulin (CRT) coding cDNA of Boophilus microplus. CRT is a calcium-binding protein involved in multiple cell functions and possibly implicated in parasites host immune system evasion. The CRT cDNA sequence and its molecular characterization are described. Sequence similarity and phylogenetic analyses indicate a close relationship to other arthropod CRT sequences. The CRT cDNA was also expressed in a procariotic system and the recombinant protein (rBmCRT) was used to raise antibodies in a rabbit. Expression analyses of the corresponding gene in different developmental stages and tissues were performed by RT-PCR and Western-blot, which indicated a ubiquitous expression of the B. microplus calreticulin gene and demonstrated its presence in saliva. Sera of tick-infested bovines suggested that this protein may not be able to induce an IgG-based humoral response in its natural host.     

The Distribution and Ecological Preferences of Boophilus microplus (Acari: Ixodidae) in Mexico

This paper focuses on the distribution of the cattle tick, Boophilus microplus, in Mexico. The study is aimed to understand the climate factors responsible of the recorded distribution that can statistically define the suitable habitat for the tick. Sites where the tick is recorded display significantly higher values of some climate variables in comparison with those where the tick is absent, namely mean monthly temperature (T) and atmospheric water vapour (W), yearly accumulated T, W and rainfall (R) (p < 0.001 for every variable), with smaller significance for the yearly sum of T/R and T/W ratios (p < 0.05). Interestingly, variables involving the Normalized Derived Vegetation Index (NDVI) do not shown statistical differences between the sites where the tick is present or absent. The best set of habitat-defining variables was integrated into a framework to assess the habitat suitability for the tick in Mexico. We used a point-to-point similarity metric to assign a classification value to a candidate site based on the proximity in environmental space of the most similar record site. A combination of 7 yearly and monthly values for temperature, rainfall and water vapour variables captured the tick distribution. Model performance, as tested with a separate set of distribution tests and defined by the AUC value, was 0.89. Causes of errors as detected with a visual comparison of both known and predicted distribution of the tick may be attributed to the use of a medium resolution, unable to capture locally important features of tick distribution, and to incomplete collections in some parts of the country.     

Efficacy of cyhalothrin and lambdacyhalothrin against Boophilus microplus (Acari: Ixodidae).

A study was conducted to determine the efficacy of cyhalothrin and lambdacyhalothrin as a whole-body spray and lambdacyhalothrin as a pour-on application on cattle infested with Boophilus microplus (Canestrini). A laboratory bioassay was also done with both spray formulations. Cattle were infested with all parasitic life stages of B. microplus, then treated with 0.007 or 0.01% (AI) concentrations of cyhalothrin as a spray; 0.005, 0.007, or 0.01% (AI) concentrations of lambdacyhalothrin as a spray, or 1% (AI) lambdacyhalothrin as a pour-on. As determined by calculations of the index of reproduction (IR), the 0.007 and 0.01% (AI) cyhalothrin provided 92.4 and 97.3% control, respectively; the 0.005, 0.007, and 0.01% (AI) lambdacyhalothrin provided 92.4, 98.2 (average of two treatments) and 99.3% control, respectively; and the lambdacyhalothrin pour-on treatment provided < 50% control. Bioassay results indicated lambdacyhalothrin to be twice as effective as cyhalothrin against B. microplus.     

Control in vitro tick (Boophilus microplus; Acari: Ixodidae) through plant extracts

Control in vitro tick (Boophilus microplus; Acari: Ixodidae) through plant extracts. Hydro-alcohol extracts of ten tropical plants were tested by the immersion method on mature ticks (Boophilus microplus Canestrini 1887). The biological evaluation was made with raw hydro alcoholic extracts (R-OH) to determine the most promissory compounds, which later were fractioned in their polar and non-polar parts. The polar parts showed a significantly inferior effect. The species that produced the highest mortality were Zizigium aromaticum, Morus alba, Piper nigrum and the mixture of Allium sativum- Z. aromaticum (all non-polar); on oviposition: M. alba, P. nigrum and the mixture of A. sativum - Z. aromaticum (all non-polar); on eclosion: P. nigrum, Z. aromaticum, Echinacea angustiofilia, Poligonum punctatum, M. alba and the mixture of A. sativum-Z. aromaticum (all non-polar): on control percentage: P. nigrum, E. angustifolia, P. punctatum and M. alba (all non-polar, except for M. alba in which both fractions showed important activity); and finally, on the percentage of oviposition inhibition: P. nigrum, E. angustifolia, P. punctatum, Gliricidia sepium and M. alba.     

Levels of natural resistance to Boophilus microplus (Acari: Ixodidae) in Carora breed bulls.

Boophilus microplus infestation is one of the most serious limitations to cattle industry in tropical regions, even though bovines show natural resistance to ticks. This resistance was evaluated in Cross-bred Carora Bulls (CCB) a tropicalized dairy breed from Venezuela. Seven CCB were experimentally infested with B. microplus larvae, "Mozo" strain, they were considered tick-naive because they had never been infested with ticks. The mean inoculum size applied on each bull was 6,477 larvae. After life cycle was completed adult female body weight (BW), egg mass weight (EW), egg hatching rate (%EH), and reproductive index (RI) were recorded. Results revealed a high variability in the levels of resistance to B. microplus. Thus, one animal showed greater resistance (Dunnett, p < 0.05) for the analyzed parameters in contrast with three non-resistant bulls. The others had moderate resistance. The trait "resistance" should be included together with other traits often used in genetic selection of cattle.     

Isolation and identification of an esterase from a Mexican strain of Boophilus microplus (Acari: Ixodidae)

A strain of Mexican Boophilus microplus (Cz) collected near Coatzacoalcos, Veracruz, Mexico, exhibits a moderate, but significant, level of permethrin resistance. Unlike other highly permethrin resistant strains, the Cz strain does not have a mutation within the sodium channel gene that results in target-site insensitivity. However, the Cz strain possesses a substantial increase in general and permethrin esterase activity relative to highly permethrin resistant and control strains suggesting the involvement of a metabolic esterase(s) in the expression of permethrin resistance. We report the isolation of a 62.8 kDa protein from Cz strain larvae that we think is the esterase previously reported as Cz EST9. In addition, internal amino acid sequence data obtained from the 62.8 kDa protein suggest that it is the gene product of a previously reported B. microplus carboxylesterase cDNA. We propose that the 62.8 kDa protein (Cz EST9) has permethrin hydrolytic activity and as a result plays an important role in Cz strain resistance to permethrin.     

Comparative inhibition kinetics for acetylcholinesterases extracted from organophosphate resistant and susceptible strains of Boophilus microplus (Acari: Ixodidae)

In this study, acetylcholinesterases (AChEs) were extracted from two Mexican Boophilus microplus strains that demonstrated resistance to the organophosphate (OP) acaricide, coumaphos, in bioassay. The rate of inhibition of the extracted AChEs by the diethyl-OP paraoxon was determined for two resistant strains and two susceptible strains of B. microplus. The time to inhibition of 50% AChE activity was approximately two-fold greater for the resistant strains. Kinetic analysis of the interaction of the resistant AChEs with paraoxon revealed reduced bimolecular reaction constants (ki). Apparent conformational changes in the AChE of the resistant strains were reflected in reduced Km and Vmax values. The bimolecular reaction constants (ki) of the resistant strains were most affected by a slower rate of enzyme phosphorylation (k2).     

Effect of selection pressure on the cholinesterase of Boophilus microplus (Acari: Ixodidae) resistant to coumaphos

The effect of selection pressure on the cholinesterase (AChE) activity of two strains of Boophilus microplus (Canestrini) resistant to coumaphos was monitored. Total AChE and protein was determined from three generations of resistant ticks and a susceptible strain. The effect of an AChE inhibitor, coroxon (the oxygen analog of coumaphos), was also determined. The resistance of the susceptible strain (Escondido) to coumaphos remained relatively unchanged throughout the study. The Tuxpan strain lost some of its resistance to coumaphos as the generations proceeded (AChE increased instead of decreased). The Tuxtla strain became more resistant to coumaphos as the generations proceeded (AChE increased).     

Effectiveness of Beauveria bassiana sensu lato strains for biological control against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in China

Owing to the need to combat the spread of acaricide-resistant ticks, the development of long-term biological control has become a hot topic for tick control. In this study, we investigated the pathogenicity of three Beauveria bassiana isolates on the engorged female Rhipicephalus (Boophilus) microplus ticks using different conidial concentrations. The results showed that B. bassiana B.bAT17 was highly pathogenic against engorged R. (B.) microplus females, resulting in lethal time (LT₅₀ and LT₉₀) of 7.14 and 9.33 days at a concentration of 10⁹ conidia/ml. R. (B.) microplus females treated with B. bassiana B.bAT17 significantly reduced the amount of ovipositioning; and most ticks died before they could begin to oviposit. Proteases and chitinases were analyzed in order to establish a screening method for identification of high virulent strains. This study has confirmed the significant pathogenic effect of entomopathogenic fungi against engorged R. (B.) microplus females in China, and further studies on the efficiency of the fungus against ticks in the field are required.     

Biological Parameters of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) Fed on Rabbits,Sheep, and Cattle

In order to determine the effect of various hosts on feeding performance of Rhipicephalus (Boophilus) microplus, we used 3 mammalian species as hosts, cattle (Qinchuan), sheep (T an), and rabbits (Japanese white rabbit) for infest-ing ticks. Five hundreds of R. microplus larvae were exposed to each animal (3 animals/host species). Tick recoveries were 11.0%, 0.47%, and 5.5% from cattle, sheep, and rabbits, respectively. The averages of tick feeding periods were not significantly different on cattle, sheep, and rabbits, 28.8, 25.3, and 26.7 days, respectively. The average weights of individual engorged female from cattle, sheep, and rabbits were 312.5, 219.1, and 130.2 mg, respectively and those of egg mass weights each to 85.0, 96.6, and 17.8 mg. The highest egg hatching rate was in the ticks from cattle (96.0%), fol-lowed by those from rabbits (83.0%) and sheep (19.2%). These data suggest that rabbits could be as an alternative host to cultivate R. microplus for evaluating vaccines and chemical and biological medicines against the tick in the laboratory, although the biological parameters of ticks were less than those from cattle.     

Molecular and Biochemical Diagnosis of Esterase-Mediated Pyrethroid Resistance in a Mexican Strain of Boophilus microplus (Acari: Ixodidae)

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. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative reproduction and nonparasitic development of Boophilus microplus and hybridized Boophilus ticks (Acari: Ixodidae) under natural field conditions in subtropical South Texas

The reproductive biology and nonparasitic development of Boophilus microplus (Canestrini) and hybridized Boophilus ticks (B. annulatus (Say) male x B. microplus female) held under natural field conditions in south Texas throughout the year were compared. Comparisons between the two types of ticks indicated that the ovipositional biology (percentage of ovipositing females and number of eggs laid) of the females favored hybrid ticks during some months and B. microplus ticks during other months. However, on a yearlong basis, there was virtually no difference in the percentage of ovipositing females or in the number of eggs deposited by hybrid females as compared to B. microplus. The duration of each nonparasitic development period (preoviposition period, incubation period of eggs, and larval longevity) showed that both types of ticks had very similar developmental and survival rates during the year. Generally the difference in duration of each of the nonparasitic parameters was <8 days, leading to a high degree of synchrony of the nonparasitic developmental rates between the two types of ticks. On the other hand, egg hatchability of hybrid ticks was consistently lower than pure-strain B. microplus throughout the year with significantly lower hatch rates occuring in April, July, August, October, and November. Thus, results obtained on percentage of ovipositing females, number of eggs laid, preoviposition period, incubation period of eggs, and larval longevity provide positive encouragement for the possible use of sterile hybrid males as a means of eliminating a native B. microplus population. In contrast, results of the egg hatchability of the two types of ticks indicate that the selective advantage afforded toB. microplus could have an adverse affect on the success of a sterile hybrid male program by making the number of hybrid ticks necessary to eliminate a native population prohibitively high.This paper reports the results of research only. Mention of a commercial or a proprietary product in this paper does not constitute an endorsement by the US Department of Agriculture.     

Susceptibility of the cattle tick Boophilus microplus (Acari: Ixodidae) to isolates of the fungus Beauveria bassiana

The susceptibility of the tick Boophilus microplus to Beauveria bassiana was evaluated by inoculating eggs, larvae and engorged females of the tick with five fungal isolates at concentrations of 10⁶, 10⁷ and 10⁸ conidia/ml. Tick eggs (0.25 g) were immersed in 1 ml of a suspension of the different conidial concentrations for 1 min. Similar exposure was performed by immersion of 2000 larvae and homogeneous groups of nine engorged females in 2 and 20 ml of conidial suspension, respectively. Treated eggs, larvae and adults were placed in an incubator at 27 ± 1 °C and relative humidity above 80% for evaluation of the fungal action. All fungal isolates applied at all conidial concentrations reduced the hatching rate of larvae from treated eggs by 1.36–65.58% and increased the mortality rate of inoculated larvae by 0.8–70.49%. In the bioassay with engorged females, oviposition period was reduced by 9.69–47.80%, egg mass weight by 4.71–53.87%, estimated reproduction by 8.3–60.62%, egg production index by 5.03–54.20%, percent larval hatching by 0.27–13.96%, and the mortality rate of treated females was increased by 96.60–100%. The reduction of the estimated reproduction obtained for the treated groups ranged from 8.37 to 64.52%. The sporulation of the pathogen on dead females ranged from 3.70 to 88.88% depending on the isolate and concentration used. Isolates AM 09, CB 7 and JAB 07 were the most effective and effectiveness increased with increasing concentrations of conidia in the suspensions.     

Molecular cloning of a phospholipid-hydroperoxide glutathione peroxidase gene from the tick, Boophilus microplus (Acari: Ixodidae)

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) enzymes are associated with cellular protection by the role they play in reducing hydroperoxides of phospholipids, thereby preventing membrane lipoperoxidation. As part of their toxic effect, some pesticides stimulate peroxidation of cellular membranes. We isolated and sequenced a PHGPx gene from the cattle tick Boophilus microplus that encodes a protein of 169 amino acids, including a TGA-encoded selenocysteine at residue 46 and active site residues Gln⁸² and Trp¹³⁵ that interact with the selenocysteine. The motif that directs the insertion of selenocysteine at the opal codon is found in the 3′-untranslated region. PHGPx sequences from pesticide-resistant and susceptible B. microplus ticks show nucleotide differences at eight positions among the strains, with five resulting in amino acid substitutions in the deduced protein sequence. Two distinct PHGPx alleles were identified in an organophosphate-resistant tick strain. Real-time PCR quantification of gene expression revealed increased PHGPx in two strains resistant to a single acaricide class. Strains resistant to two or more classes showed a reduction in PHGPx.     

In vitro activities of plant extracts from the Brazilian Cerrado and Pantanal against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

A total of 73 ethanol extracts from different anatomical parts of 44 plant species belonging to 24 families, native to the Mid-Western region of Brazil, were assessed in vitro for their effect on the reproductive cycle of engorged females of the cattle tick Rhipicephalus (Boophilus) microplus, using the adult immersion test. All extracts were evaluated at the concentration of 0.2 % and, among the extracts tested, the one obtained from the fruits of Guarea kunthiana (Meliaceae) proved to be highly efficacious, showing 99.1 % of product effectiveness. Extracts from other three species were shown to be moderately active, namely Nymphaea amazonum trunk (Nymphaeaceae) [51.7 %], Strychnos pseudoquina trunk (Loganiaceae) [8.0 %] and Ocotea lancifolia leaves (Lauraceae) [34.5 %], while the remaining extracts were shown to be weakly active or inactive. This is the first report on the bioactivity of these species on egg production by engorged females of R. microplus.     

Inheritance of resistance to flumethrin in the Mexican Aldama strain of the cattle tick Boophilus microplus (Acari: Ixodidae)

The objective of this study was to evaluate the inheritance mode of resistance to flumethrin in the Mexican Aldama Boophilus microplus strain. Two Mexican strains were used, the Chiapas susceptible (SS), and the Aldama flumethrin-resistant from Tamaulipas. Six steers weighing ca. 250 kg were randomly assigned for each of six crosses: the susceptible (SS), resistant (RR), and the F1 (RS, SR) reciprocal crosses and F2 (RS x RS and SR x SR). The reciprocal crosses were made to evaluate maternal and sex linkage effects. Bioassays tested resistant and susceptible larvae along with their hybrid F1 and F2 progeny against a series of concentrations of flumethrin (0, 0.0075, 0.00150, 0.00300, 0.00600 and 0.01200 microg/g). To test the single-gene hypothesis of resistance, a nonparametric line-cross test proposed by Collins was used. The bioassay data were subjected to probit analysis and the resistance factor and effective dominance obtained. Results of this study indicated that inheritance for flumethrin resistance in the Aldama strain was autosomal and controlled for more than one gene. The F1 and F2 larvae had similar lower resistant factor (RF 2.8-4.5) while the resistant Aldama strain was 21-fold higher (RF 81.8) than the mean of the F1 and F2. The extent of flumethrin resistance in the Aldama B. microplus strain depended upon the concentration of the pesticide used. Resistance was almost dominant at the lowest dose while almost completely recessive at the highest dose. Maternal effects were shown for egg-mass. These results shown here indicate more than one gene basis of flumethrin resistance in B. microplus ticks are present. Therefore it is necessary to locate and understand the major loci for elucidate the mechanism of resistance and improve the ability to track and delay the evolution of resistance.