Amitraz Resistance Research Articles

A new mutation in the octopamine receptor associated with amitraz resistance in Varroa destructor

A new mutation in the octopamine receptor associated with amitraz resistance in <scp><i>Varroa destructor</i></scp>

Amitraz Resistance in French Varroa Mite Populations-More Complex Than a Single-Nucleotide Polymorphism.

Resistance against amitraz in Varroa mite populations has become a subject of interest in recent years due to the increasing reports of the reduced field efficacy of amitraz treatments, especially from some beekeepers in France and the United States. The loss of amitraz as a reliable tool to effectively reduce Varroa mite infestation in the field could severely worsen the position of beekeepers in the fight to keep Varroa infestation rates in their colonies at low levels. In this publication, we present data from French apiaries, collected in the years 2020 and 2021. These data include the field efficacy of an authorized amitraz-based Varroa treatment (Apivar® ,Véto-pharma, France) and the results of laboratory sensitivity assays of Varroa mites exposed to the reference LC90 concentration of amitraz. In addition, a total of 240 Varroa mites from Eastern, Central, and Southern regions in France that were previously classified as either "sensitive" or "resistant" to amitraz in a laboratory sensitivity assay were genotyped. The genetic analyses of mite samples are focused on the β-adrenergic-like octopamine receptor, which is considered as the main target site for amitraz in Varroa mites. Special attention was paid to a single-nucleotide polymorphism (SNP) at position 260 of the ORβ-2R-L gene that was previously associated to amitraz resistance in French Varroa mites, Varroa. Our findings confirm that amitraz resistance occurs in patches or "islands of resistance", with a less severe reduction in treatment efficacy compared to pyrethroid resistance or coumaphos resistance in Varroa mites. The results of our genetic analyses of Varroa mites call into question the hypothesis of the SNP at position 260 of the ORβ-2R-L gene being directly responsible for amitraz resistance development.

Novel polymorphisms in the octopamine receptor gene of amitraz resistant population of Rhipicephalus sanguineus sensu lato, in south India

The brown dog tick or Rhipicephalus sanguineus sensu lato is an ixodid tick, responsible for the dissemination of pathogens that cause canine infectious diseases besides inflicting the direct effects of tick bite. The hot humid climate of Kerala, a south Indian state, is favorable for propagation of tick vectors and acaricides are the main stay of tick control. Though the resistance against synthetic pyrethroids is reported among these species, the status of amitraz resistance in R. sanguineus s. l. in the country is uncertain due to the lack of molecular characterisation data and scarce literature reports. Hence the present study was focused on the phenotypic detection and preliminary genotypic characterisation of amitraz resistance in the R. sanguineus s. l. A modified larval packet test (LPT) on a susceptible isolate was performed to determine the discriminating dose (DD). Further LPT-DD on 35 tick isolates was carried out to detect amitraz resistance robustly, along with that full dose response bioassays on the resistant isolates were performed. The results indicated that amitraz resistance is prevalent with 49 per cent of the samples being resistant. Amplification of exon 3 of octopamine receptor gene from both the susceptible and resistant larval isolates was carried out. Amplicons of ten pooled amitraz susceptible and ten pooled amitraz resistant representative samples were sequenced and analysed, unveiling a total of three novel non-synonymous mutations in the partial coding region at positions V32A, N41D and V58I in phenotypically resistant larval DNA samples. In silico analysis by homology modelling and molecular docking of the mutated and unmutated receptors showed that these mutations had reduced the binding affinity to amitraz. However, lack of mutations in the octopamine receptor gene in three of the pooled low order resistant R. sanguineus s. l. larval samples could be suggestive of other mechanisms associated with amitraz resistance in the region. Hence, further association studies should be carried out to confirm the association of these mutations with target insensitivity in R. sanguineus s. l. ticks, along with exploring the status of metabolic resistance and other mechanisms of resistance.

Experimental parameters affecting the outcomes of amitraz resistance testing in Varroa destructor

Honey bee colony health is impaired by a large variety of biotic and abiotic factors. The severe impacts of Varroa destructor and viruses associated with it on honey bees are undeniable and universal. Beekeepers often utilize miticides to mitigate V. destructor populations, but resistance to many widely used synthetic miticides such as amitraz has developed. The Apivar® resistance test has proven to be a valuable tool to monitor for amitraz resistance in V. destructor. However, a more thorough understanding of the effects that changes in testing parameters have on the outcome of the Apivar® resistance is critical for an accurate and comparable interpretation of the results. In this project, the effects of temperature, product age, product exposure surface area, and container reuse on the Apivar® resistance test were studied. High temperature significantly increased control mortality while low temperatures significantly increased Apivar® resistance. The increased Apivar® resistance at low temperature was due to reduced amitraz toxicity at low temperature. There was no association of Apivar® age or manufacturing batch on the outcome of the Apivar® resistance test. The maintenance of Apivar® efficacy with age is likely because the amitraz breakdown product N‘-(2,4-dimethylphenyl)-N-methylformamidine (DPMF) is as toxic as amitraz. The rate and efficacy of the Apivar® resistance test were higher with a larger surface area of Apivar® strip. Reused testing containers or Apivar® strips from colony application yielded reduced efficacy and consistency. This research shows the standard conditions needed to produce comparable data from the Apivar® resistance test. Implications for amitraz resistance monitoring are discussed.

Contrasting roles of cytochrome P450s in amitraz and chlorfenapyr resistance in the crop pest Tetranychus urticae

The molecular mechanisms of amitraz and chlorfenapyr resistance remain only poorly understood for major agricultural pests and vectors of human diseases. This study focusses on a multi-resistant field strain of the crop pest Tetranychus urticae, which could be readily selected in the laboratory to high levels of amitraz and chlorfenapyr resistance. Toxicity experiments using tralopyril, the active toxophore of chlorfenapyr, suggested decreased activation as a likely mechanism underlying resistance. Starting from the same parental strain, transcriptome profiling revealed that a cluster of detoxifying genes was upregulated after amitraz selection, but unexpectedly downregulated after chlorfenapyr selection. Further functional validation associated the upregulation of CYP392A16 with amitraz metabolism and the downregulation of CYP392D8 with reduced activation of chlorfenapyr to tralopyril. Genetic mapping (QTL analysis by BSA) was conducted in an attempt to unravel the genetic mechanisms of expression variation and resistance. This revealed that chlorfenapyr resistance was associated with a single QTL, while 3 QTLs were uncovered for amitraz resistance. Together with the observed contrasting gene expression patterns, we argue that transcriptional regulators most likely underly the distinct expression profiles associated with resistance, but these await further functional validation.

Acaricide Resistance Development in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) Populations against Amitraz and Deltamethrin on Communal Farms of the King Sabata Dalindyebo Municipality, South Africa.

Chemical acaricides are widely used to control ticks and tick-borne pathogens in cattle. However, prolonged and indiscriminate use of these chemicals inevitably leads to the selection of resistant ticks. In-vitro bioassays (adult and larval immersion tests) were conducted to assess amitraz and deltamethrin resistance in Rhipicephalus (Boophilus) microplus populations from communal farms of the King Sabata Dalindyebo municipality of South Africa. Data generated on percentage inhibition of oviposition (%IO) revealed that all the tick populations assessed showed resistance (%IO ≤ 95%) to at least one of the acaricides. All six tick populations assessed for efficacy (%IO ≥ 95%) at the DD) with deltamethrin were resistant (%IO ≤ 95%) and only one of the six tick populations assessed for efficacy with amitraz was susceptible. Based on the resistance ratios (RR), the adult immersion test detected amitraz and deltamethrin resistance in three (RR ranging from 2.30 to 3.21) and five (RR ranging from 4.10 to 14.59) of the six tick populations, respectively. With the larval immersion test, deltamethrin and amitraz resistance (larval mortality < 90% at the DD) was detected in all four and three of four R. (B.) microplus populations assessed, respectively. These data are critical for the design of an effective and sustainable tick control strategy on the communal farms.

Assessment of cypermethrin and amitraz resistance and molecular profiling of voltage-gated sodium channel and octopamine tyramine genes of Rhipicephalus microplus.

Control of ticks and tick-borne pathogens is a priority for human and animal health. Livestock-holders extensively rely on acaricide applications for tick control. Different groups of acaricides including cypermethrin and amitraz have been consistently used in Pakistan. There has been a gap in understanding the susceptibility or resistance of Rhipicephalus microplus, the most prevalent tick in Pakistan, to acaricides. The present study aimed to molecularly characterize cypermethrin and amitraz targeted genes such as voltage-gated sodium channel (VGSC) and octopamine tyramine (OCT/Tyr) of R. microplus ticks in Khyber Pakhtunkhwa (KP), Pakistan to monitor the acaricides resistance. Tick specimens were collected from cattle and buffaloes in northern (Chitral, Shangla, Swat, Dir, and Buner), central (Peshawar, Mardan, Charsadda, Swabi, and Nowshera), and southern districts (Kohat, Karak, Lakki Marwat, Tank, and Dera Ismail Khan) of KP, Pakistan. Different concentrations of commercially available cypermethrin (10%) and amitraz (12.5%) were prepared for in vitro larval immersion tests (LIT). In LIT, the average mortality rate of immersed larvae was recorded that was increased gradually with an increase in the concentration of specific acaricide. The larvae's highest mortality rates (94.5% and 79.5%) were observed at 100-ppm of cypermethrin and amitraz, respectively. A subset of 82 R. microplus ticks was subjected to extract genomic DNA, followed by PCR to amplify partial fragments of VGSC (domain-II) and OCT/Tyr genes. The BLAST results of the consensus sequence of VGSC gene (domain-II) showed 100% identity with the acaricides susceptible tick sequence from the United States (reference sequence). Obtained identical sequences of OCT/Tyr genes showed maximum identity (94-100%) with the identical sequences reported from Australia (reference sequence), India, Brazil, Philippines, USA, South Africa, and China. Thirteen single nucleotide polymorphisms (10 synonymous and three non-synonymous) were observed at various positions of partial OCT/Tyr gene fragments. The SNP at position A-22-C (T-8-P) in OCT/Tyr gene has been linked to amitraz resistance in R. microplus ticks. Molecular analysis and LIT bioassay's findings indicate the availability of resistant R. microplus ticks in the KP region. To our understanding, this is the first preliminary study to monitor cypermethrin and amitraz resistance via molecular profiling of cypermethrin and amitraz targeted genes (VGSC and OCT/Tyr) in combination with in vitro bioassays (LIT) in R. microplus ticks from Pakistan.

Confirmation of the Y215H mutation in the β2 -octopamine receptor in Varroa destructor is associated with contemporary cases of amitraz resistance in the United States.

The parasitic mite, Varroa destructor (Anderson and Trueman), is a leading cause of honey bee colony losses around the world. Application of miticides such as amitraz are often the primary method of Varroa control in commercial beekeeping operations in the United States. It is likely that excessive and exclusive amitraz application has led to the development of amitraz resistance in Varroa. A mutation of tyrosine at amino acid position 215 to histidine (Y215H) in the β2 -octopamine receptor was identified in putatively amitraz-resistant Varroa in the United States. This research investigated the presence of the Y215H mutation in quantitatively confirmed amitraz-resistant Varroa from the United States. There was a strong association of susceptible and resistant phenotypes with the corresponding susceptible and resistant genotypes respectively, and vice versa. The resistance bioassay may understate resistance levels because of the influence of environmental conditions on the outcome of the test, whereby Varroa with an amitraz-resistant genotype may appear with a susceptible phenotype. Confirmation of the Y215H mutation in the β2 -octopamine receptor of amitraz-resistant Varroa encourages the development and validation of low-cost, high-throughput genotyping protocols to assess amitraz resistance. Resistance monitoring via genotyping will allow for large-scale passive monitoring to accurately determine the prevalence of amitraz resistance rather than directed sampling of apiaries with known resistance issues. Genotyping of Varroa for amitraz resistance early in the beekeeping season may predict late-season resistance at the colony level and provide beekeepers with enough time to develop an effective Varroa management strategy. © 2023 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Adult immersion test for assessment of amitraz resistance in Rhipicephalus sanguineus ticks

Ticks are obligate blood sucking ectoparasites recognised globally as vectors of various pathogens, next to mosquitoes. The present study is conducted on brown dog ticks collected from naturally infested dogs in and around Thrissur district. Adult immersion test was performed and the parameters such as engorged tick weight, egg mass weight as well as tick mortality were recorded to derive the reproductive indices, percentage inhibition of oviposition and percentage mortalities. Non-linear regression analysis of the dose response data yielded the LC50 values at 95% confidence intervals and, the slopes and regression coefficients of the different parameters were also derived. The Resistance factors (RF) were calculated to categorise resistance levels into I, II, III and IV. Resistance to amitraz was detected at level II in tick isolates collected from Choolissery and Mundur, at level III and level IV in isolates collected from Mannuthy and Paravattani, respectively. Two isolates collected from Mannuthy were found to be susceptible. One way ANOVA of resistance levels revealed that the resistance levels III and IV were significantly different from that of susceptible and resistance level II. This is the first report on detection of amitraz resistance levels in dog ticks from south India calling for proper implementation of tick control strategies to prevent spread of amitraz resistance.

Molecular Characterization of Octopamine/Tyramine Receptor Gene of Amitraz-Resistant Rhipicephalus (Boophilus) decoloratus Ticks from Uganda.

We previously reported the emergence of amitraz-resistant Rhipicephalus (Boophilus) decoloratus ticks in the western region of Uganda. This study characterized the octopamine/tyramine receptor gene (OCT/Tyr) of amitraz-resistant and -susceptible R. (B.) decoloratus ticks from four regions of Uganda. The OCT/Tyr gene was amplified from genomic DNA of 17 R. (B.) decoloratus larval populations of known susceptibility to amitraz. The amplicons were purified, cloned and sequenced to determine mutations in the partial coding region of the OCT/Tyr gene. The amplified R. (B.) decoloratus OCT/Tyr gene was 91-100% identical to the R. (B.) microplus OCT/Tyr gene. Up to 24 single nucleotide polymorphisms (SNPs) were found in the OCT/Tyr gene from ticks obtained from high acaricide pressure areas, compared to 8 from the low acaricide pressure areas. A total of eight amino acid mutations were recorded in the partial OCT/Tyr gene from ticks from the western region, and four of them were associated with amitraz-resistant tick populations. The amino acid mutations M1G, L16F, D41G and V72A were associated with phenotypic resistance to amitraz with no specific pattern. Phylogenetic analysis revealed that the OCT/Tyr gene sequence from this study clustered into two distinct groups that separated the genotype from high acaricide pressure areas from the susceptible populations. In conclusion, this study is the first to characterize the R. (B.) decoloratus OCT/Tyr receptor gene and reports four novel amino acid mutations associated with phenotypic amitraz resistance in Uganda. However, lack of mutations in the ORF of the OCT/Tyr gene fragment for some of the amitraz-resistant R. (B.) decoloratus ticks could suggest that other mechanisms of resistance may be responsible for amitraz resistance, hence the need for further investigation.

Monitoring the resistance of Rhipicephalus microplus to amitraz, flumethrin, coumaphos, and ivermectin on cattle farms in Mexico.

The prevalence, resistance ratios and factor associated with Rhipicephalu microplus populations resistant to amitraz, flumethrin, coumaphos, and ivermectina (IVM) in Mexico were studied. Field tick populations were collected from 54 farms in 15 different states of Mexico. The dose-response bioassays were carried out using the larval immersion test (amitraz and IVM) and the modified larval packet test (flumethrin and coumaphos) against R. microplus. Mortality data were subjected to probit analysis to calculate lethal concentrations at 50%. A logistic regression model was used to evaluate the relation between resistance and possible associated factors. The phenotype was defined as high resistant (HR), low resistant (LR) or susceptible (S). The overall prevalence of cattle farms with R. microplus resistant to coumaphos, amitraz, flumethrin and IVM were 25.9, 46.2, 31.5 and 68.5%, respectively. For coumaphos, 74.1, 22.2, and 3.7% were classified as S, LR, and HR, respectively, whereas, for amitraz, 53.7, 24.1, and 22.2% of phenotypes were S, LR, and HR, respectively, for flumethrin 68.5, 14.8, and 16.7% were S, LR and HR respectively, and for IVM, 31.5, 46.3, and 22.2% were S, LR, and HR, respectively. We identified that cattle farms without acaricide rotation program (OR: 7.66, CL95%: 1.70-34.47, P: 0.008) had a higher probability of developing R. microplus resistant to amitraz. We concluded that amitraz and IVM resistance in R. microplus is frequent, but mainly at a low level in cattle farms of Mexico. Cattle farms without acaricide rotation program had higher probability of developing R. microplus resistant to amitraz.

Modelling the impact of Apivar treatment on a Varroa mite population and the influence of resistance.

Varroa destructor is a parasite of honeybees. It causes biological damage leading to the colony collapse in the absence of treatment. In recent years, acaricide resistance has emerged in Varroa mites, leading to a decrease in treatment efficacy. We modelled the action of Apivar (amitraz) treatment, using three input parameters: treatment duration, treatment period, and daily mortality due to the treatment. The output parameters were cumulative mite mortality during treatment, the residual number of Varroa mites, and treatment efficacy, expressed as a percentage. The model was validated by monitoring efficacy in the field, in 36 treated hives. According to the model, treatment in the absence of brood is optimal. For a long period without egg laying during the winter, an initial infestation of 100 mites and a start date for treatment of August 7, a minimal treatment efficacy of 98.8% is required for stabilization of the mite population for year to year. More effective treatment is associated with lower cumulative numbers of dead Varroa mites over the entire treatment period. Thus, the total number of dead mites observed during the monitoring of field efficacy provides information about more than just the initial level of colony infestation. The proportion of resistant mites can be modelized by a decrease of daily mortality rate influencing treatment efficacy. Management of the initial Varroa mite infestation of the colony by the beekeeper can compensate for the decrease in treatment efficacy for resistance thresholds of up to 40% of resistant mites. Treatment efficacy depends on several parameters, including initial level of infestation, treatment period and the presence of acaricide resistance. Amitraz resistance may lead to treatment failure, even if the beekeeper is able to keep initial infestation rates low. © 2021 Society of Chemical Industry.

Adult bioassay based Amitraz resistance status in Rhipicephalus microplus populations of Punjab, India

Adult bioassay based Amitraz resistance status in Rhipicephalus microplus populations of Punjab, India

Whole genome sequencing and bulked segregant analysis suggest a new mechanism of amitraz resistance in the citrus red mite, Panonychus citri (Acari: Tetranychidae).

Amitraz is a broad-spectrum insecticide/acaricide for the control of aphids, psyllids, ticks and mites. Current evidence suggests that ticks and phytophagous mites have developed strong resistance to amitraz. Previous studies have shown that multiple mechanisms are associated with amitraz resistance in ticks, but very few reports have involved Panonychus citri. We therefore used whole genome sequencing and bulked segregant analysis (BSA) to identify the mechanism underlying P. citri's resistance to amitraz. High-quality assembly of the whole P. citri genome was completed, resulting in a genome of approximately 83.97 Mb and a contig N50 of approximately 1.81 Mb. Gene structure predictions revealed 11 577 genes, of which 10940 genes were annotated. Trait-associated regions in the genome were mapped with bulked segregant analysis and 38 candidate SNPs were obtained, of which T752C had the strongest correlation with the resistant trait, located at the 5' untranslated region (UTR) of the β-2R adrenergic-like octopamine receptor gene. The mutation resulted in the formation of a short hairpin loop structure in mRNA and gene expression was down-regulated by more than 50% in the amitraz-resistant strain. Validation of the T752C mutation in field populations of P. citri found that the correlation between the resistance ratio and the base mutation was 94.40%. Our results suggest that this 5' UTR mutation of the β-2R octopamine receptor gene, confers amitraz resistance in P. citri. This discovery provides a new explanation for the mechanism of pest resistance: base mutations in the 5' untranslated region of target gene may regulate the susceptibility of pests to pesticides.

Mechanisms of amitraz resistance in a Rhipicephalus microplus strain from southern Brazil

Amitraz is one of the most used acaricides for the control of ticks of domestic animals, however, extensive use of this active ingredient has favored the development of resistant populations of Rhipicephalus microplus worldwide. The possible mechanisms of metabolic and/or target-site alterations mechanisms of amitraz resistance were investigated in a Brazilian field population of R. microplus (São Gabriel strain). Bioassays with the synergists piperonylbutoxide, triphenylphosphate and diethyl-maleate were used to evaluate the metabolic mechanisms involved. Target-site insensitivity was investigated by amplification and sequencing of a fragment of the octopamine/tyramine (OCT/TYR) receptor gene. Piperonylbutoxide synergism (synergism ratio = 2.8) indicated the participation of the P450 pathway in the detoxification of amitraz. Previously reported single nucleotide polymorphisms that confer amino acid changes in the OCT/TYR receptor, threonine to proline (T8P) and leucine to serine (L22S), were found in the amitraz-resistant strain but not in the susceptible reference strain. The results suggest that amitraz resistance in the studied strain is multi-factorial and may result from cytochrome P450 detoxification and mutations in octopamine receptors.

Genotyping amitraz resistance profiles in Rhipicephalus microplus Canestrini (Acari: Ixodidae) ticks from Punjab, India

Acaricide resistance is one of the greatest threats to sustainable and effective control of vector ticks worldwide. The amitraz resistance status in cattle tick, Rhipicephalus microplus populations collected from 18 districts of Punjab in north-western India were characterized using bioassay and molecular assays. The modified larval packet test was used and the resistance factors (RF) against amitraz for the field populations were in the range of 0.36–4.85, indicating level I resistance status in ten populations. Characterization of a partial segment of the octopamine/tyramine (OCT/Tyr) receptor gene of R. microplus field populations from Punjab revealed a total of 18 nucleotide substitutions in the coding region out of which 5 were non-synonymous substitutions. Three of these non-synonymous substitutions (T8P, V15I and A20 T) were earlier reported in American and South African populations of R. microplus. Among the two single nucleotide polymorphisms (A22C-T8P; T65C-L22S) potentially linked to amitraz resistance in American, South African and Zimbabwean resistant populations, only the T8P substitution was recorded from the Barnala population. The PCR-RFLP assay using EciI restriction enzyme was used for genotyping of the larvae as homozygous resistant (RR), homozygous susceptible (SS) and heterozygous (SR). Genotyping of 514 larval DNA samples from 18 field populations revealed 92.8 % larval population as SR and the remaining 7.2 % as RR genotypes. The percentage of resistant alleles in the tick populations was 53.6 (range 50.0–57.2) indicating its moderate distribution in the region. The present study is the pioneer report establishing the hypothesis that amitraz-resistance is recessively inherited and heterozygous individuals show phenotypic susceptibility to the drug in the Indian tick populations.

Resistance of ticks on cattle to amitraz in Zimbabwe.

Resistance of one host and three host ticks on cattle to amitraz was studied using samples from five diptanks in the Domboshawa Communal Land Area of Zimbabwe. A random tick profile and a questionnaire survey on the tick control practices of the area were also carried out. Engorged Rhipicephalus (Boophilus) decoloratus, Rhipicephalus appendiculatus and Amblyomma hebraeum females were randomly collected from cattle presented for dipping at the 5 diptanks and were allowed to oviposit separately at T: 28°C and RH: 85-95%. Larvae obtained were tested for resistance against various amitraz concentrations (1-0.0078125%) using the Larval Packet Test (LPT) and were compared with susceptible reference strains of R. (B.) decoloratus (Makuti strain, 2017), R. appendiculatus (Lake Chivero strain, 2015) and A. hebraeum (Lake Mutirikwi strain, 2017). The most abundant tick species were R. (B.) decoloratus (27.2%), Hyalomma rufipes (20.0%), H. truncatum (16.0%), R. appendiculatus (12.0%) and R. evertsi evertsi (11.9%). Amblyomma hebraeum (8.6%) and A. variegatum (1.8%) were the least common in the collection; this suggests that they were not well established in Domboshawa. Low amitraz resistance (RL = I) was detected only in R. (B.) decoloratus at 2 of the 5 diptanks. In the future, decentralised tick control due to inadequate and inconsistent supply of acaricides could introduce a number of factors which could contribute towards resistance development.

Molecular screening of cattle ticks, tick-borne pathogens and amitraz resistance in ticks of Santo Domingo de los Tsáchilas province in Ecuador

The province of Santo Domingo de los Tsáchilas in Ecuador is a strategic place for cattle production and trade. The lack of knowledge about tick species, tick control and tick-borne diseases in Ecuador prompted this study with the goal of identifying the cattle-tick species and tick-borne agents present in the area and molecularly determining the potential acaricide resistance to amitraz of the major cattle tick species. Eighty-four cattle farms were visited and in 88 % of them, cattle were infested with ticks. Additionally, 24 historical samples from other surrounding Ecuadorian provinces, were screened as well. Besides morphological keys, PCR-RFLP MspI was used to confirm the presence of the Rhipicephalus ticks. The tick samples were also screened for tick-borne agents using PCR-RFLP BseDI and Hhal tests to identify circulating Babesia sp. and Anaplasma spp. Furthermore, the PCR-RFLP EciI technique was used to identify the amitraz resistance gene in populations of Rhipicephalus microplus in the province. Pooled testing was used to determine prevalence at individual-tick level. The presence of R. microplus and Amblyomma cajennense sensu lato (s.l.) ticks was found in 83 % and 21 % of the cattle farms respectively, showing R. microplus is widespread in the province of Santo Domingo de los Tsáchilas. Regarding tick-borne agents, only Anaplasma marginale was observed in 50 % of the visited farms of the province, while about 27 % of the ticks tested positive according to estimations from the data of the tick pools. The presence of Babesia bigemina was only confirmed in samples collected outside the province. The amitraz resistance allele in R. microplus was found in 62 % of the farms, but the percentage of farms with cattle ticks completely resistant to this acaricide was low (2%). The findings of this study should prompt cattle producers and animal health authorities to monitor control strategies, which address the management of resistant tick populations and the epidemiologically-unstable areas of tick-borne diseases.

A point mutation in the β-adrenergic-like octopamine receptor: possible association with amitraz resistance.

Amitraz is a unique formamidine-class acaricide/insecticide that effectively controls ticks, mites, and insect pests. However, the recent emergence of amitraz-resistant cattle ticks is a serious problem that requires an urgent solution. A nonsynonymous single nucleotide polymorphism (A181T) leading to an amino acid substitution (I61F) in the β-adrenergic-like (β-AL) octopamine receptor (OAR) of amitraz-resistant southern cattle ticks (Rhipicephalus microplus) (RmβAOR) was proposed to be a cause of the amitraz resistance. However, it remains unclear whether this substitution exerts any functional effect on the action of amitraz. To make this clear, the functional role of this mutation was examined using an orthologous OAR (BmOAR2) from the silkworm (Bombyx mori). Both amitraz and its metabolite N2 -(2,4-dimethylphenyl)-N1 -methyformamidine (DPMF) elevated intracellular cyclic AMP levels as orthosteric OAR agonists in HEK-293 cells stably expressing BmOAR2. The I45F mutant of BmOAR2 (equivalent to I61F in RmβAOR) was generated and tested for its sensitivity to amitraz and DPMF. The assay result showed that the I45F mutation reduces the potency of DPMF to a level similar to that of the endogenous agonist (R)-OA in wild-type BmOAR2. The amino acid substitution found in the first transmembrane segment of RmβAOR most likely causes target-site insensitivity to DPMF, which might contribute to the resistance of R. microplus to amitraz. This needs to be further confirmed using RmβAOR. © 2020 Society of Chemical Industry.

Multiple acaricide-resistant Rhipicephalus microplus in the semi-arid region of Paraíba State, Brazil

Rhipicephalus microplus is responsible for major economic losses in cattle ranching in Brazil, and the development of multiple acaricide resistance is a major concern to control this ectoparasite. To determine the resistance of R. microplus to acaricides in 26 ranches in the semi-arid region of Paraíba State, Brazil, larval assays were performed with cypermethrin, chlorpyriphos, amitraz, ivermectin and fipronil and resistance was observed in all locations. The frequency of populations resistant to cypermethrin, chlorpyriphos, amitraz and ivermectin was, respectively, 96 %, 72 %, 83 % and 92 % while none was resistant to fipronil. Resistance to one, two, three and four acaricides was recorded, respectively, in 4 %, 15 %, 31 % and 50 % of the farms. Errors in the control management of R. microplus determined the development of resistance to acaricides in all evaluated properties pointing towards development of new strategies for its control. In order to control multiple acaricide-resistant cattle tick populations in this area it is recommended to apply suppressive treatments with fipronil and/ or fluazuron.