Rhipicephalus microplus is the most important tick in veterinary medicine, given its repercussions on animal production. The principal strategy to avoid adverse effects associated with R. microplus is the chemical control of tick populations through organosynthetic acaricides. Therefore, monitoring susceptibility to acaricides is paramount in any control program. This study aimed to analyze the resistance status of 2 populations of R. microplus from northeastern Mexico to the organochlorine (OC) lindane, organophosphates (OP) coumaphos, chlorfenvinphos, diazinon, and chlorpyrifos, and the synthetic pyrethroids (SPs) flumethrin, deltamethrin, and cypermethrin. Discriminating doses (DD) of each acaricide were used in the larval packet bioassay (LPT). Additionally, the presence of the knockdown resistance (kdr) mutation T2134A associated with pyrethroid resistance was evaluated using allele-specific polymerase chain reaction (PCR). The populations of R. microplus showed a high frequency of resistance to SP, with mortality rates of less than 5%; they also showed resistance to the OPs (diazinon and chlorpyrifos) with mortality rates ranging from 1.29% to 34.62%; meanwhile, they were susceptible to coumaphos and chlorfenvinphos. Mortality rates higher than 66% were observed for lindane, indicating susceptibility. The mutant allele of the kdr mutation T2134A was detected in 75% and 100% of the pools analyzed. The populations studied presented a highly resistant profile to pyrethroids, with the presence of the kdr mutant allele A2134. The susceptibility to the organophosphates such as coumaphos and chlorfenvinphos of R. microplus from northeastern Mexico should be noted.
Rhipicephalus microplus (Canestrini) represents the most important ectoparasite problem for cattle production in tropical and subtropical regions worldwide (Rodríguez-Vivas et al. 2018). It generates economic losses estimated between 13.9 and 18.7 billion dollars annually worldwide (Betancur Hurtado and Giraldo-Ríos 2019). These losses are generated by direct effects such as damages to production assets and indirect consequences caused by the R. microplus–mediated transmission of Babesia bigemina (Smith et Kilborne), Babesia bovis Babes and Anaplasma marginale Theiler (Solorio-Rivera et al. 1999, Rodríguez-Vivas et al. 2005, de la Fuente et al. 2007). Controlling tick populations using synthetic acaricides is the principal strategy to avoid adverse impacts of R. microplus. However, the high intensity of their use in tick management has led to tick resistance to all major classes of acaricides (Rodríguez-Vivas et al. 2006a, 2006b; Perez-Cogollo et al. 2010).
Resistance to acaricides has been recorded in more than 2,000 populations of R. microplus to different toxicants, such as deltamethrin, cypermethrin, flumethrin, and many others (Dzemo et al. 2022). In Mexico, R. microplus has developed resistance to permethrin, coumaphos, chlorfenvinphos, diazinon, chlorpyrifos, flumethrin, deltamethrin, cypermethrin, amitraz, ivermectin, and fipronil (Guerrero et al. 2002; Rodríguez-Vivas et al. 2006a, 2013; Perez-Cogollo et al. 2010; Fernández-Salas et al. 2012a, 2012b; Miller et al. 2013; Rodríguez-Vivas et al. 2014).
Four mechanisms have been determined to reduce the efficacy of pesticides in arthropods: changes in insect behavior, thickening of the insect cuticle, increased activity of detoxifying enzymes, and modification of the target site (Dang et al. 2017, Zalucki and Furlong 2017, Ndiath et al. 2019). The increased metabolism of insecticides through elevated enzyme activity and decreased sensitivity of the target site are the 2 major mechanisms reported in ticks (Janadaree Bandara and Parakrama Karunaratne 2017). An example of the latter is the knockdown resistance (kdr) mutations, nonsynonymous point mutations in the para gene for transmembrane voltage-gated sodium channel proteins, which cause reduced binding of pyrethroids and dichlorodiphenyltrichloroethane (DDT) to their target site. In R. microplus, 5 kdr mutations (T170C, C190A, G215 T, T2134A, and T2134C) have been demonstrated to be associated with pyrethroid resistance (He et al. 1999, Morgan et al. 2009, Jonsson et al. 2010, Stone et al. 2014, Villar et al. 2020); whereas other nonsynonymous mutations C148T, G184C, C190G, and synonymous mutations C189A and C2130T did not appear to be associated with resistance, or their involvement in conferring resistance has been found to merit further investigation (Stone et al. 2014). In Mexico, the G184C, C190A, and T2134A mutations have been associated with maintaining muscle contractility in R. microplus when exposed to cypermethrin, suggesting their participation in the resistance to this pyrethroid (Cossío-Bayúgar et al. 2020).
The development of resistance is a complex and dynamic process and depends on many factors. To better understand the development of resistance, we need to have both a basic understanding of phenotype and good knowledge of the mechanisms that cause the resistance. Although the dose–response bioassays remain the gold standard method of quantifying acaricide resistance (Rodríguez-Vivas et al. 2018), the discriminating-dose bioassay has been used to encourage the continuous field level monitoring of the status of acaricide resistance (FAO 2004, Gupta et al. 2022). The present study aimed to assess the resistance status of various acaricides in 2 tick populations from northeastern Mexico. We further investigated the presence of the T2134A kdr mutation.
MATERIALS AND METHODS
Live engorged R. microplus ticks were carefully removed from cattle and handled in the laboratory following the breeding recommendations proposed by the FAO (2004) to produce progeny. Ticks were collected in the Northeastern Regional Investigation Center from Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP; 22°34′1.995600″N, 98°9′47.1924″W) and the Don Enrique ranch (22°58′00″N, 98°04′00″), both in the municipality of Aldama, Tamaulipas, Mexico. Collected ticks were kept under a controlled temperature between 27 ± 2°C and 85 ± 5% relative humidity (RH) in a biochemical oxygen demand incubator (Riossa, model E33, Mexico City, Mexico) to induce oviposition. Egg masses were weighed and stored in a 10 ml assay tube until larvae were obtained and used for the larval packet test (LPT).
Larval packet test
The susceptibility to the organochlorine (OC) lindane, organophosphates (OPs), and synthetic pyrethroids (SPs) was evaluated through the larval packet test (Stone and Haydock 1962), using discriminating doses (DD) (FAO 2004). The LPT assay was conducted using 14–21 day old unfed larvae. Tests were performed with Whatman No. 1 filter papers of 7.5 cm × 8.5 cm with trichloroethylene as a diluent and olive oil as the fixer. Each packet was impregnated individually, using DD of lindane (0.1 mg/ml), coumaphos (0.2 mg/ml), chlorfenvinphos (0.2 mg/ml), diazinon (0.08 mg/ml), chlorpyrifos (0.02 mg/ml), flumethrin (0.01 mg/ml), deltamethrin (0.09 mg/ml), and cypermethrin (0.5 mg/ml), as well as trichloroethylene as a control (FAO 2004). All insecticides were technical grade obtained from Chem Service® (Chem Service, West Chester, PA). All packets were incubated at 28 ± 2°C and 85 ± 5% RH for 24 h. Six replicates were performed for each acaricide in each population. On average, each replicate consisted of 116 larvae exposed to the DD in LPT assays. After incubation, live and dead larvae were counted to calculate the mortality rate.
The mortality rate was calculated by dividing the total number of dead larvae by the total number of exposed larvae in each packet and multiplying it by 100. Since mortality was found in the control group, the mortality rates were corrected following the Abbott's formula (Abbott 1925). We considered the mortality rate cutoff proposed by Fragoso and Soberanes (2001), where mortality rates below 60% indicated a resistant population. A 1-way analysis of variance (ANOVA) was conducted to test for differences in the mortality rate between all acaricides in the INIFAP population. The ANOVA was followed by an honestly significant difference (HSD) Tukey post hoc test. For the Don Enrique population, differences in the mortality rates between acaricides were analyzed using the Kruskal–Wallis test and the Wilcoxon sum rank test, since the assumptions of normality and homogeneity of variances were not met. All statistical tests were conducted in R (version 4.2.2), and α was set to 0.05.
Detection of T2134A kdr mutation
We investigated the presence of the kdr mutation T2134A using allele-specific PCR (Guerrero et al. 2001). The DNA extraction was performed through the modified salt technique described by Coen et al. (1982). Briefly, 8 pools from each location composed of 5 larvae of R. microplus were macerated individually in 50 μl lysis buffer. The DNA was precipitated using 200 μl of 100% ethanol and incubated for 2 h at −20°C. Two wash steps with 75% ethanol were followed to eliminate contaminants. Finally, the DNA was resuspended in molecular-grade water. The PCR reactions were performed in a final volume of 25 μl containing: 200 ng of genomic DNA, 10 μl of Taq polymerase mix (Promega, Madison, WI), and 25 pM of each primer: Wild type antisense 5′-GAATAGATTCAAGGTGAA-3′, resistant allele, 5′-GAATAGATTCAAGGTGAT-3′, universal antisense 5′-TTGTTCATTGAAATTGTCGA-3′, and universal sense 5′-CTAACATCTACATGTACC-3′ (Guerrero et al. 2001). Negative control using water instead of DNA as a template was used. The PCR reactions were subject to the following amplification conditions: initial denaturalization of 94°C for 4 min, followed by 36 cycles of 94°C for 1 min, 53°C for 45 sec, and 72°C for 30 sec, and a final extension of 72°C for 6 min. The PCR products were evaluated by a 2% agarose gel electrophoresis.
Resistance of Rhipicephalus microplus to organosynthetic compounds
The LPT assays showed that both populations of R. microplus presented mortality rates greater than 60% for the OC lindane and the OPs chlorfenvinphos and coumaphos, thus resulting in susceptibility to these chemicals (Table 1). The ANOVA showed statistically significant differences in the mean mortality rates between all tested acaricides in the INIFAP population (F = 150.6, df = 7, P < 0.0001). The HSD test revealed that the mortality rates obtained for all SP and chlorpyrifos were significantly lower (P < 0.05) than that of the OC lindane and the OPs coumaphos, chlorfenvinphos, and diazinon. The mortality rates for diazinon were significantly higher than those of pyrethroids and chlorpyrifos, but significantly lower than chlorfenvinphos, coumaphos, and lindane (P < 0.05) (Fig. 1).
For the Don Enrique population, the Kruskal–Wallis test showed statistically significant differences in the mortality rates between all tested acaricides (H = 39.175, df = 7, P < 0.0001). The mortality rates of diazinon, chlorpyrifos, and all pyrethroids, were significantly lower than those of lindane, coumaphos, and chlorfenvinphos (P < 0.05), as the pairwise Wilcoxon test revealed. No statistically significant difference was observed in the mortality rates between lindane, coumaphos, and chlorfenvinphos (Fig. 1B).
Detection of T2134A mutation in Rhipicephalus microplus
We identified the nucleotidic modification (T to A) in the 2,134 position that promotes an aminoacidic change from phenylalanine to isoleucine in both collection sites by detecting a 68-bp fragment. Eight pools of each population were evaluated by amplifying a region of the voltage-gated sodium channel (VGSC), using allele-specific primers. Our results indicate that 100% of the pools from the INIFAP population carried the mutation, while 75% of the analyzed pools from the Don Enrique population carried the mutant allele.
Routine monitoring of acaricide resistance in natural tick populations helps to detect resistance early and improve the effectiveness of operational control strategies. For this purpose, the Food and Agriculture Organization (FAO 2004) recommends specific bioassay techniques to test resistance in ticks: the larval packet test (LPT), the larval immersion test (LIT), the larval tarsal test (LTT), and the adult immersion test (AIT). However, regardless of the technique used, phenotypic resistance can be expressed in 2 ways, as the proportion of ticks that are not killed by a given dose of acaricide (discriminating dose or DD) or as the ratio of the dose of acaricide required to kill a given proportion of a test population (i.e., 50%, 90%, or 99%) compared with a susceptible reference strain, that is the resistance ratio (RR) (Rodríguez-Vivas et al. 2012, Guerrero et al. 2014). Using DD of the acaricides allowed us to determine that R. microplus from northeastern Mexico resulted in ticks susceptible to the OC lindane and the OPs chlorfenvinphos and coumaphos, but showed resistance to the OPs diazinon, chlorpyrifos, and the SPs flumethrin, deltamethrin, and cypermethrin. In this study, we use the LPT technique with DD of technical grade acaricides, which, although it has its limitations due to the selection of adequate DD (Johnson et al. 2007), represented a valuable tool when the number of samples to be processed is high, helping monitor changes in susceptibility in tick populations over time.
The organophosphates are globally used for R. microplus control; in Mexico, this group of insecticides was used intensively between 1974 and 1984 during the national campaign for Boophilus tick eradication (Trapapa 1989). The OPs used during this period included coumaphos, chlorpyriphos, chlorfenvinphos, diazinon, and ethion. The 1st case of resistance to OP was detected in R. microplus from southern Mexico in 1983. The strain of ticks established from that location demonstrated 10- to 14-fold resistance (compared to the reference susceptible strain) to coumaphos, chlorpyriphos, and ethion (Aguirre and Santamaría 1986). Resistance to OPs soon became widespread in the central, eastern, and southern regions of Mexico (Fragoso et al. 1995). Currently, the list of OP acaricides used for the control of R. microplus in Mexico continues to include coumaphos and chlorpyrifos, as well as diazinon and clorfenvinfos, alone or in a mixture with SP (Alonso-Díaz and Fernández-Salas 2022), so the monitoring of susceptibility to these molecules continues to be of great relevance in decision-making in the control of R. microplus. In our study, R. microplus populations were susceptible to OP coumaphos; however, studies like Li et al. (2003) showed resistance to this acaricide, with RR50 values ranking 1.07- to 10.09-fold in populations of R. microplus from eastern Mexico, including 2 populations from northeastern Mexico. In this study, the authors also showed resistance to the OP diazinon, even at higher levels than coumaphos, with RR50 values ranking 1.88- to 34.44-fold. The authors also showed that resistance to coumaphos was likely to be conferred by the cytP450-mediated detoxification mechanism and suggested a possible role for glutathione S-transferases in diazinon detoxification. Our study confirmed resistance to diazinon in R. microplus from Don Enrique and INIFAP, with mean mortality rates of 5.18 and 34.62, respectively. The susceptibility observed in our study for the OPs coumaphos and chlorfenvinphos and resistance to diazinon and chlorpyrifos may be due to different resistance mechanisms (i.e., detoxifying enzymes) associated with each insecticide, as demonstrated by Li et al. (2003).
Similarly, populations of R. microplus with distinct phenotypic resistance patterns might show different expression levels of genes involved in detoxification. Cossío-Bayúgar et al. (2008) determined that strains of R. microplus resistant to organophosphates, pyrethroids, and amidines had lower expression levels of cytP450 than populations only resistant to pyrethroids. Other studies regarding in vitro glutathione S-transferase (GST) inhibition by acaricides concluded that some miticides, such as ethion, amitraz, chlorpyrifos, DDT, cypermethrin, diazinon, deltamethrin, and flumethrin, inhibit GST activity. However, ivermectin does not inhibit this enzyme; contrastingly, coumaphos activates GST (da Silva Vaz et al. 2004). However, this is a limitation in our study, since the enzymatic resistance mechanisms in the studied populations were not characterized.
Resistance to the OPs chlorpyrifos, coumaphos, and diazinon has been previously confirmed in R. microplus from the Mexican Pacific coast (Olivares-Pérez et al. 2011) using the LPT technique, and the DD of acaricides resulted in mortality rates of 36.7% for chlorpyrifos, 25.3% for coumaphos, and 18.6% for diazinon. However, resistance was not generalized in all the farms sampled, since the prevalence [No. of ranches with acaricide-resistant ticks/No. total ranches sampled (100)] was 31.1%, 48.3%, and 82.2% to chlorpyrifos, coumaphos, and diazinon, respectively.
More recent studies in populations of R. microplus from the Mexican Gulf coast show susceptibility to OPs, specifically to chlorpyrifos; however, the evaluation was carried out with a formulation containing 60 g dichlorvos + 20 g chlorpyrifos (Higa et al. 2020). Similar situations have been reported in different Brazilian regions by Higa et al. (2016), where the susceptibility of R. microplus to this formulation (dichlorvos 60 g + 20 g chlorpyrifos) was verified, even though resistance to chlorpyrifos has been widely recognized (Higa et al. 2015).
Regarding resistance to OC lindane, there is only 1 report in Mexico of R. microplus from the Gulf Coast. Although Mexico has adhered to international treaties for the elimination of lindane since 2010, in 2018, the Federal Commission for Protection against Sanitary Risks (COFEPRIS, the Spanish acronym for that Federal Commission) confirmed the existence of 4 registered products of this insecticide in the country (NHRC 2018), so the validity of its use in the country is uncertain. In our study, we did not find resistance to this acaricide, since the mean mortality rate in the R. microplus populations analyzed was >60%; however, it is important to consider the history of lindane use due to the risk that it could represent in the development of cross-resistance with other acaricides, as shown by Castro-Janer et al. (2015), who found cross-resistance between lindane and fipronil in R. microplus from Uruguay and Brazil.
Pyrethroids have been extensively used for decades to control livestock ectoparasites in Mexico, with permethrin being the 1st SP (Rosario-Cruz et al. 2005) used. These insecticides were introduced into Mexico in 1986 to alleviate OPs resistance problems in ticks, but 7 years later (1993), the 1st reports of SP resistance were described, using larval package dose discriminant assays; by this time, most of the populations screened were already resistant to organophosphate acaricides (Ortiz et al. 1995). In the present study, both populations of R. microplus were resistant to SP flumethrin, deltamethrin, and cypermethrin, with mean mortality rates of less than 5%.
Numerous studies indicate the occurrence of different populations of R. microplus from Mexico resistant to SP. Of 42 documented reports of resistance in R. microplus from Mexico, 33% (14/42) correspond to SP, among which flumethrin, deltamethrin, and cypermethrin stand out (Rodriguez-Molano et al. 2020).
Given the low mortality rates (<5%) of flumethrin, deltamethrin, and cypermethrin in both populations, we investigated the presence of the T2134A single nucleotide polymorphism (SNP) in the gene encoding the para-sodium channel, the target site of SP. Our results indicate that this mutation is present in both tick populations, as revealed by the AS-PCR. Notably, pyrethroid resistance in R. microplus is now globally expanded (Yessinou et al. 2016, Dzemo et al. 2022), and it has been correlated with 5 SNPs, including T170C, G184C, C190A, and C190G found in the II domain of the sodium channel, along with the SNP T2134A in the III domain (He et al. 1999, Morgan et al. 2009, Jonsson et al. 2010, Stone et al. 2014, Villar et al. 2020). The other 5 SNPs (3 nonsynonymous: C148T, G184C, and C190G; and 2 synonymous: C189A and C2130) require further studies to understand their participation in pyrethroid resistance (Kumar et al. 2020). The SNP T2134A appears to be restricted geographically to the United States, detected in R. microplus collected in Texas (Miller et al. 2007, Stone et al. 2014) and found in acaricide-selected individuals originally collected from several Mexican states, including Yucatan (Rosario-Cruz et al. 2005, 2009; Rodríguez-Vivas et al. 2012); Nuevo León (Li et al. 2007); Colima (Chen et al. 2009), and Tamaulipas (Chen et al. 2009, Lovis et al. 2012). It is important to mention that this investigation is limited, as we only determined the presence of SNP T2134A. However, other studies aiming to estimate the frequency of the A2134 mutant allele are necessary to link this SNP to pyrethroid resistance. Finally, results must be interpreted cautiously, considering that other alternative resistance mechanisms could play a role in the observed resistance patterns (Waldman et al. 2023).
We thank Grecia Valencia de Haro and Lizeth Guadalupe Perez Mata for their collaboration in this research.
Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Biologicas, Av. Universidad s/n Cd. Universitaria, San Nicolas de los Garza, Nuevo León, 66455, Mexico.
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, CIR-Noreste, C. E. Río Bravo, Tamaulipas, Mexico.