Resistance profile of Rhipicephalus (Boophilus) microplus to diazinon and cypermethrin and first report of sodium channel mutation - domain III S6 - T2134A, in field samples from the state of São Paulo, Brazil

Rhipicephalus (Boophilus) microplus is one of the parasites that has an impact on livestock farming in Brazil. Bioassays using the larval packet test (LPT) impregnated with cypermethrin and diazinon at different concentrations were performed to characterize phenotypic resistance (resistance level, RL), molecular resistance (mutation in domain III - S6 T2134A) and enzymatic metabolism of diazinon and cypermethrin in some populations of R. microplus that were collected in different regions of the state of São Paulo. Among the 40 farms analyzed, 18 of them presented resistance factors for cypermethrin, with RL I (53%) and RL II (47%). Mutation of domain III T2134A was found for the first time in Brazil, on seven farms. Heterozygous larvae were found on six of them and resistant homozygous larvae on four. No differences (p>0.05) in enzymatic activity of α-esterase and β-esterase were found in tests with live and dead larvae at a cypermethrin concentration of 409.6μg/cm 2 . Ninety percent of the farms showed resistance to the active agent diazinon: RL I in 6%, RL II in 30% and RL III in 64%. There were significant differences (p<0.05) in enzymatic activity at the highest concentration (3.2μg/cm 2 ).

Chemical treatment is the main means of controlling R. microplus in cattle.The main chemical groups used are organophosphates, pyrethroids, formamidines, macrocyclic lactones, phenylpyrazoles and fluazuron.Resistance of ticks to these groups is widespread in Brazil.In the state of São Paulo, all these acaricide classes are used (Lovis, 2013;Higa et al., 2016).Fourteen years ago, the products most used by farmers for treating cattle consisted of associations of pyrethroids and organophosphates.This was especially so in the Paraíba valley region, which is considered to be one of the largest milk-producing regions of the state of São Paulo (Mendes et al., 2007).
Metabolic resistance is conferred by a metabolic process in which insects are able to detoxify or break down the toxin faster than susceptible insects.It is known that some enzymatic systems may be involved in biotransformation and excretion of insecticides in resistant strains and may have higher levels of expression over a broad spectrum of activity relating to detoxification (Guidelines…, 2004).
Four families of enzymes are associated with insecticide metabolism: carboxylesterases (CCEs), glutathione S-transferases (GSTs), uridine 5'-diphospho-glucuronosyltransferases (UDP-glucuronosyltransferase, UGTs) and cytochrome P450s (CYP450).These have been implicated in conferring resistance to one or more classes of insecticides (Rosario-Cruz et al., 2009;Cossío-Bayogar et al., 2018;Feyereisen et al., 2015).Brito et al. (2017) used a polymerase chain reaction (qPCR) to quantify the enzymes acetylcholinesterase and esterase in diazinonresistant R. microplus strains that were obtained from Pirajuí and Presidente Médice, RO, Brazil.Chigure et al. (2017) studied the correlation between resistance and functional enzymes in R. microplus populations in India.Several research groups have focused on characterization of acaricide resistance through bioassays and development of new diagnostic methodologies, such as molecular tests to quickly detect resistance with the objective of directing correct management of acaricides.
As already known, non-metabolic resistance occurs at acaricide target sites through nucleotide substitutions in the gene, which lead to insensitivity of the target site to acaricide.According to Narahashi (2002), the target site for pyrethroids is the sodium channel that is present in the cells of the central and peripheral nervous system.This targeting results in prolonged opening of individual channels, which causes paralysis and death of these arthropods.Kumar et al. (2020) presented research data from different locations around the world and reported that at least ten single-point mutations (single nucleotide polymorphisms, SNPs) that replaced the sodium channel gene had been identified.These authors considered that five nonsynonymous mutations out of these ten (T170C, C190A, G215 T, T2134A and T2134C) were clearly associated with resistance to pyrethroids.On the other hand, five other SNPs (nonsynonymous mutations C148 T, G184C and C190 G and synonyms C189A and C2130 T) did not seem to be related to resistance.In Brazil, the C190A mutation in domain II S4-5 (resulting from substitution of amino acids from leucine to isoleucine) was the only mutation reported in tick populations that were resistant to pyrethroids.
The present study was carried out to characterize phenotypic and molecular resistance (mutation in domain III -S6 T2134A) and enzymatic metabolism of diazinon and cypermethrin in populations of ticks collected in different regions of the state of São Paulo.In the laboratory, the female ticks were kept in an incubation chamber at 28°C and 80% humidity for oviposition to take place.After this, the eggs were packed into tubes (5.3cm high by 2.4cm in diameter) that were closed with dampened cotton fiber, to await hatching of the larvae.These larvae were later used to perform resistance tests using the technique Stone and Haydock (1962).
Approximately one hundred larvae of R. microplus were transferred to each packet using a brush.The packets were then sealed with clips and incubated in an incubation chamber at 28°C and 80% relative humidity.These tests were performed in triplicate.After 24 hours, the dead and living individuals were counted.Larvae that were paralyzed or only moving their appendages, without the ability to walk, were considered dead.
The mortality data from the larval packet test were analyzed using the "probits" module of the POLO-PC software (Leora Software, 1987).The resistance level (RL) classification was calculated as described by Mendes et al. (2007), which distributes the resistance levels according to the chart below: Presence or absence of the T2134A mutation of domain III was ascertained using individual larvae of samples P10, P11, P14, P15, P16, P19, P20, P27, P36 and P37 (sample size "n" given on Table 2), in accordance with the protocol of Guerrero et al. (2001).Genomic DNA was extracted using the Quick-DNA™ Miniprep Plus extraction kit (D4069; Zymo Research ® ), following the manufacturer's guidelines.
The enzymatic activity was tested with five surviving larvae and five dead larvae that were removed from the filter paper impregnated with the highest concentration of each product.In relation to diazinon, this test was performed on fifteen RL III farms, while for cypermethrin it was performed on three RL I and six RL II farms.
The enzymatic assays were carried out in accordance with the micro burette method.To determine the total protein concentration, two reagents were used: copper sulfate 6mmol/L (reagent 1) and NaOH 1.15mol/L (reagent 2).Each sample was homogenized in two replicates, using 500μL of each reagent, 995μL of distilled water and 5μL of the sample.The standard used was bovine serum albumin at a concentration of 50g/L.Absorbance measurements at 570nm were made in a Femto 600 plus spectrophotometer and the results were expressed in g/L.
The enzymatic activity of the esterase was determined through addition of α and β-naphthyl.In each well, 20μL of the homogenate supernatant (for both α and β-esterase) was added to 250μL of α/β-25 naphthyl acetate solution dissolved in 24.75mL of 20 mM sodium phosphate buffer (pH 7.2).The reaction was incubated at room temperature for 30 minutes.Then, 50μL of Fast Blue B solution (0.045g of Fast Blue B) was added to 4.5mL of distilled water that had been added to a solution of 15mL of 5% SDS.The reaction was incubated at room temperature for another 5 minutes and then 200μL of α/β-naphthyl acetate/sodium phosphate and another 50μL of Fast Blue were added.Absorbance readings were made at 570nm.Analysis of variance was performed to determine any differences in enzymatic activity between live and dead larvae for each active agent.The Tukey test was used to compare the means, taking the significance level to be 5%.

RESULTS
The active agents used on each farm and the resistance data on the products cypermethrin and diazinon are presented in Tables 1 and 3. Use of pyrethroids and organophosphates was cited by 52.5% of the farm owners, and 20% reported using unassociated pyrethroids.Other chemical groups were mentioned with the following frequencies: 15% formamidine, 12.5% fluazuron, 10% macrocyclic lactones, 7.5% homeopathy, 5% unassociated organophosphates and 2.5% phenylpyrazoles; and 17.5% did not mention any product.
Table 1 presents test results using cypermethrin.The resistance factor was evaluated using LC 50 from farm P2 (LC 50 : 1.08), used as susceptible.Among the 40 farms analyzed, eighteen of them presented resistance factors ranging from 1.58 to 28.8, with RL I (10 farms) and RL II (8 farms).Out of these eighteen resistant samples, only five of them did not report use of cypermethrin (alone or in association with another agent) for treating animals against R. microplus.These resistance levels indicate if the acaricide resistance is still building up or if it is already established.
Mutation of domain III T2134A of the ten samples of R. microplus analyzed (Table 2) was identified on seven farms (60% heterozygous and 40% homozygous).In the P10 sample, with resistance ratio of 10.3, 87.5% of the larvae presented heterozygous resistance.The P19 sample, with resistance ratio of 28.2, showed larvae with homozygous resistance for mutation III T2134A (Figure 3).Graphs of the enzymatic activity of α-esterase and β-esterase, tested with live and dead larvae at a cypermethrin concentration of 409.6μg/cm 2 , from samples P10, P11, P14, P15, P16, P19, P20, P27, P36 and P37, did not show any significant difference between the two groups (p>0.05)(Figures 1 and 2). Figure 2. Evaluation of the enzymatic activity of α-esterase and β-esterase in R. microplus larvae (populations from P5 to P40) exposed to diazinon.Control = larvae that were not exposed to organophosphates.

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Table 3 presents the results from R. microplus tests with the active agent diazinon.90% of the farms presented resistance, taking the P3 sample as the susceptibility standard (LC 50 : 0.03).The RL percentages found were 6% with RL I, 30% with RL II and 64% with RL III.It was observed that the farms with RL II and III presented percentages of organophosphate use of 46.6% and 69.5%, respectively, in association with another active agent, for treating cattle.The results regarding enzymatic P34, P37, P39 and P40) demonstrated significant differences (p<0.05) between the groups of live and dead larvae.

DISCUSSION
Use of chemicals against R. microplus has been considered the most effective and economical way to control it for decades.However, continuous and uncontrolled use of acaricides has led to emergence of strains that are resistant to most of the compounds available on the market, according to Higa et al. (2016).
In general, development of pest resistance to insecticides depends on the quantity and frequency of their application.In the present study, reports by the owners regarding the commercial products used revealed that most of the producers were using acaricides composed of associations of organophosphates and pyrethroids.Among these farms, 89% showed resistance to diazinon and 11% to cypermethrin.The low frequency of resistance to cypermethrin was probably related to the fact that it was used together with organophosphates in the same product, thus suggesting that organophosphate was more effective than pyrethroids when used in combination.
As shown in the results from the present study, resistance of R. microplus to cypermethrin was found in 18 of the 40 farms analyzed but it was seen that the resistance values remained low.The hypothesis of high resistance to pyrethroids in this study was discarded through the unexpected results of high susceptibility among the samples analyzed.
The resistance to acaricides observed in the present study contradicts data reported by Higa et al. (2016) and Albuquerque et al. (2010).Those authors found that R. microplus strains in the state of São Paulo were resistant to products composed only of pyrethroids and thus showed that pyrethroids had low efficacy in field populations.Lovis et al. (2013) showed that there were high levels of resistance to cypermethrin in the state of São Paulo, with resistance ratio ranging from 8.0 to 309.3.Their values are discordant with our results.Klafke et al. (2017) evaluated R. microplus samples in Rio Grande do Sul using a diagnostic dose and found that resistance to cypermethrin was present in 98.08% of the 104 samples analyzed.
The results suggest that pyrethroids have reasonable efficacy against R. microplus strains in the state of São Paulo State.One hypothesis to be considered is that the quality control on the commercial products tested may have failed.Products may also have been inadequately stored on these farms.The test on technical cypermethrin in this study caused high mortality among the larvae.
The site of action of pyrethroids is the sodium channel of arthropods, and five mutations associated with resistance were described in detail by Kumar et al. (2020).Five other nonsynonymous mutations were also found, but without any safe correlation in relation to the pyrethroid-resistant phenotype.Among these mutations, the only one reported so far in Brazil was C190A (domain II S4-5), notified by Arq.Bras.Med.Vet.Zootec., v.74, n.3, p.473-482, 2022 Andreotti et al. (2011), Domingues et al. (2012) and Lovis et al. 2013.In the present study, seven samples of R microplus (resistance factor from 3.39 to 28.2) were found to have mutation T2134A in domain III S6 (F1550I: replacement of a phenylalanine by an isoleucine).This is the first report of this mutation in Brazil.It has previously been cited in tick strains that were highly resistant to pyrethroids, in Mexico and the United States (Rodríguez-Vivas et al., 2014;Stone et al., 2014;Kumar et al., 2020).The absence of a direct correlation between resistant phenotypes and the genotypes found can be explained by the presence of other mutations for which no detection tests have been performed.The results from the present study open up two possibilities: either new trials should be implemented or questioning about the influence of other factors arises.
The phenotypes found in the larval packet test results not only may be explained by the T2134A mutation, but also may be consequences of four other mutations that have already been described as associated with resistance to pyrethroids, as reported by Kumar et al. (2020).Consideration should also be given to the metabolic capacity of R. microplus relating to phenotype variations.Occurrences of sensitive genotypes with resistant phenotypes, which would suggest that catalytic sites of certain enzymes may have higher metabolic detoxification capabilities, were reported in studies by Cossio-Bayugar et al. (2009) andMiranda et al. (2009).
Several studies have reported resistance to organophosphates in R. microplus in Brazil, as described by Mendes et al. (2011), Domingues et al. (2012), Raynal et al. (2013) and Reck et al. (2014).The results obtained for diazinon in phenotypic tests corroborated the findings of these previous studies.Thus, it can be affirmed that the situation of resistance to organophosphates oscillates among farms is a consequence of their history of use of acaricides.Animal treatment failures also contribute to this situation (Lovis et al., 2013) Regarding the activity of esterase against live and dead larvae, it was evident that the P19 sample, which was the one with the highest resistance factor (28.2) for cypermethrin, showed the highest esterase activity level.This confirms what was reported by Lovis et al. (2013), i.e., that this activity in ticks is often related to development of resistance to pyrethroids.In the enzymatic profiles detected for the larval samples tested in this study with diazinon, it was observed that increased αand β-esterase levels in live larvae showed a positive correlation with resistance factors.Similarly, the metabolic relationship of the enzyme in the resistance process was demonstrated by Miller et al. (2008) and Chigure et al. (2017).The latter authors confirmed that there was a positive correlation between higher enzymatic activity of esterase and occurrences of resistance to diazinon in R. microplus samples in Mexico.This paper presents the first report of the T2134A mutation (F1550I) in a R. microplus population in Brazil.These resistance data regarding cypermethrin and diazinon form useful information for future decision-making, considering the frequent use of acaricide products based on organophosphate compounds and their associations in different regions of the state of São Paulo.

Figure 1 .
Figure1.Evaluation of enzymatic activity of α-esterase and β-esterase in R. microplus larvae (populations from P5 to P40) exposed to cypermethrin.Control = larvae that were not exposed to cypermethrin.

Figure 3 .
Figure 3. PCR amplification of 13 samples from the P19 farm.The picture shows the results of samples 7 through 13 amplified by the primer pair specific for the sensitive (227I + 221S) or resistant allele (227I + 221R).

Table 1 .
Characterization of the phenotype of resistance and susceptibility to cypermethrin in a population of Rhipicephalus (Boophilus) microplus larvae in the state of São Paulo

Table 2 .
Frequency of mutation of domain III T2134A R. microplus samples from some regions of São Paulo, Brazil

Table 3 .
Characterization of the phenotype of resistance and susceptibility to diazinon in a population of Rhipicephalus (Boophilus) microplus larvae in the state of São Paulo