BR112017010065A2 - method for detecting a genetic polymorphism associated with susceptibility or resistance of a tick, c-dna isolated nucleic acid molecule, amplified polynucleotide, use of an isolated nucleic acid molecule or amplified polynucleotide, use of genetic markers and kit for determination of the susceptibility of a tick to amitraz - Google Patents

Abstract

The invention provides a method for detecting a genetic polymorphism associated with susceptibility or resistance of an ectoparasite to an acaricide, the method comprising the step of screening an ectoparasite DNA sample for the presence of one or more markers in the octopamine / receptor gene. tyramine.

Description

Method for Detecting a Genetic Polymorphism Associated with SUSCETIBILITY OR STRENGTH OF A TICK, Amplified POLYNUCLEOTIDE, Use of an Isolated Nucleic Acid Molecule or AN AMPLIFIED POLYNUCLEOTIDE, USE OF GENETIC MARKERS AND Kit for Determining the Susceptibility of a Tick to a Tick

Field of Invention [0001] This invention relates to the control of ectoparasites. More particularly, the invention relates to a method of identifying resistance to amitraz in an ectoparasite, for polymorphisms / genetic markers associated with such resistance and for nucleotide sequences useful for detecting such resistance markers.

History of the Invention [0002] The Rhipicephalus microplus tick (form of Boophilus microplus) is a widely invasive ectoparasite of great economic importance due to the negative effect it has on agricultural livestock on a global scale. The diseases transmitted by the tick (babesiosis and anaplasmosis) transmitted by R. microplus are alarming since they reduce the quality of livestock. In sub-Saharan Africa, cattle represent a greater source of meat and milk, but this region of the world is severely affected by the tick Rhipicephalus microplus. The main method for tick control is the use of chemical mites, notably amitraz, which was implemented in the 1990s after resistance to other mites. However, the effectiveness of chemical control is hindered by an increase in the frequency of mutant resistance alleles for amitraz in tick populations. At present, the only way to assess resistance to amitraz is by testing the larval pack, but this technique is time-consuming and not particularly cost-effective.

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2/42 [0003] Rhipicephalus microplus ticks are hematophagous ectoparasites of veterinary importance and are capable of parasitizing a variety of hosts, although cattle are their primary preference (Walker et al. 2003). These ticks are specialists in transmitting a variety of tick-borne diseases to cattle, most notably Babesia bovis, which causes Asian babesiosis or red water (Walker et al. 2003; Horak, Golezardv, Uvs 2007). The lack of effective tick control strategies and management programs results in a serious economic burden, threatening the sustainability of the livestock industry in South Africa and globally. The use of chemical acaricides is still the most preferred method for tick control, but it has become less effective due to the emergence of resistance. So far, resistance has been reported for all major classes of acaricides, including synthetic pyrethroids (He et al. 1999; Aguilar-Tipacamú et al. 2008; Chen et al. 2009; Morgan et al. 2009; Jonsson et al. 2010a), organophosphates and carbamates (Guerrero, Pruett, Li 2003; Li, Davey, George 2005; Guerrero, Lovis, Martins 2012), formamidines (Soberanes et al. 2002; Fernández-Salasa, RodríguezVivas, Alonso-Díaza 2012; Guerrero, Lovis, Martins 2012) and macrocyclic lactones (Pohl et al. 2011; Fernández-Salasa, Rodríguez-Vivas, AlonsoDíaza 2012; Fernández-Salasa et al. 2012). Resistance to acaricides was attributed to insensitivity to the destination, as well as metabolic detoxification, depending on the mode of action of the acaricide.

[0004] Rhipicephalus microplus ticks have acquired the ability to avoid the toxic effects of chemical acaricides by developing different resistance mechanisms. The cuticle that surrounds the tick, which reduces the acaricide's access to the internal environment of the tick's body, provides resistance to penetration. However, other investigations into this type of resistance in R. microplus have not been reported since 1983 (Schnitzerling, Nolan, Hughes 1983). An additional mechanism of resistance common in arthropods is destination insensitivity. This adaptive mechanism

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3/42 involves changing the drug's destination site at the DNA level by changing the wild-type allele to a mutant form, which makes the acaricidal treatment ineffective. Finally, metabolic resistance to acaricide treatment involves the increased ability to detoxify or sequester the acaricide. This involves increasing common detoxification enzymes including cytochrome P450s, esterases and glutathione-S-transferases (Guerrero, Lovis, Martins 2012). All of the mechanisms mentioned above have been shown to play a vital role in the tick's resistance to chemical acaricides.

[0005] Amitraz is a formamidine acaricide, which is used extensively for tick control in South Africa. The destination location for amitraz in R. microplus has not yet been defined, which ultimately delays any further development with regarding screening tests for diagnosis. It has been proposed that monoamine oxidase, alpha-2-adrenoceptors and the octopamine receptor are good candidates for potential target sites, with the latter being the most likely in ticks (Jonsson, Hope 2007). Amitraz is thought to be a potential agonist of the octopaminergic system located in the synaganglion tick. It has been shown that in the presence of amitraz, the octopamine receptor is activated and its over-stimulation at synapses has lethal effects on the tick (Booth 1989; Lees, Bowman 2007). Octopaminergic receptors have been classified into three distinct classes namely α-adrenergic (aOCT), β-adrenergic (βΟΟΤ), and octopamine / tyramine (OCT / Tyr) or tyramaminergic (Evans, Maqueira 2005).

[0006] Resistance to amitraz is complex and it is suggested to be of a multigenic nature, involving recessive inheritance of resistance alleles (Li et al. 2004; Li et al. 2005; Fraqoso-Sanchez et al. 2011). So far, mechanisms of adequate resistance against amitraz for R. microplus have not been shown, but several of these mechanisms have been suggested. Li et al. (2004) illustrated, through synergistic studies with enzyme inhibitors, that metabolic detoxification plays a role in resistance to amitraz.

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4/42

However, these results were variable across different strains of R. microplus, and the overall contributions of detoxification enzymes were difficult to assess. It was confirmed that the Pesqueria Mexicana tick strain transmits metabolic resistance to amitraz by increasing glutathione-Stransferase (Saldivar et al. 2008). It has also been suggested that destination site insensitivity could perhaps be the main mechanism of resistance to amitraz. Unfortunately, conclusive studies to illustrate this mechanism have not been successful (Fraqoso-Sanchez et al. 2011; Guerrero, Lovis, Martins 2012).

[0007] Baxter, Barker (1999) sequenced the putative octopamine receptor of an Australian strain resistant to amitraz and susceptible to R. microplus. Sequence analysis revealed no differentiation between the two. It was later proposed that two unique nucleotide polymorphisms (SNPs) at the octopamine receptor were linked to resistance to amitraz (Chen, He, Davev 2007). However, these SNPs were inferred only by the sequence alignment between a susceptible Australian strain, the susceptible American Gonzalez strain and the resistant strain Santa Luiza (Chen, He, Davev 2007). Sequencing the octopamine receptor gene for these strains revealed 37 SNPs, of which nine were non-synonymous substitutions. Seven of these were attributed to geographical differences between the strains, while the remaining two SNPs were potentially linked to resistance to amitraz. These two SNPs occur at position 8 of the amino acid (threonine to proline) and 22 (leucine to serine) of the octopamine receptor protein (Chen, He, Davev 2007). Recent studies then indicated that the previous work was probably carried out on an OCT / Tyr type receptor, instead of the native octopamine receptor (Corley, Piper, Jonsson 2012). This consequently led to the discovery of a SNP (I61F) in the R. microplus βadrenergic octopamine receptor (Corley et al. 2013). This particular SNP was located only in central Queensland (Australia) and absent in the north and southeast regions, thus minimizing its probability of being the reason

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5/42 major for resistance to amitraz in Australia. Currently, the only way to effectively assess resistance to amitraz is through packet larvae tests (LPTs) (Stone, Havdock 1962); however, these trials are limited in practicality, as they take approximately six weeks to obtain the results.

[0008] All references to tick DNA sequences and single nucleotide polymorphisms (SNPs) refer to the nucleotide sequences of the coding region only, positions of non-coding regions are excluded.

References [0009] Agapow, P-M, A Burt. 2001. Indices of multilocus linkage disequilibrium. Molecular ecology notes 1: 101-102.

[0010] Aguilar-Tipacamú, G, RJ Miller, R Hernández-Ortiz, Rl RodriguezVivas, C Vásquez-Peláez, Z García-Vázquez, F Olvera-Valencia, R RosarioCruz. 2008. Inheritance of pyrethroid resistance and a sodium channel gene mutation in the cattle tick Boophilus microplus. Parasitol Res 103: 633-639.

[0011] Aljanabi, SM, I Martinez. 1997. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Research 25: 4692-4693.

[0012] Balfanz, S, T Strünker, S Frings, A Baumann. 2005. A family of octopamine receptors that specifically induce cyclic AMP production or Ca2 + release in Drosophila melanogaster. Journal of Neurochemistry 93: 440-451.

[0013] Baxter, GD, SC Barker. 1999. Isolation of a cDNA for an octopamine-like, G-protein coupled receptor from the cattle tick, Boophilus microplus. . Insect Biochemistry and Molecular Biology 29: 461-467.

[0014] Booth, TF. 1989. Effects of biogenic amines and adrenergic drugs on oviposition in the cattle tick Boophilus: evidence for octopaminergic innervation of the oviduct. Experimental and Applied Acarology 7: 259-266.

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6/42 [0015] Chen, AC, H He, RB Davey. 2007. Mutations in a putative octopamine receptor gene in amitraz-resistant cattle ticks. Veterinary Parasitology 148: 379-383.

[0016] Chen, AC, H He, KB Temeyer, S Jones, P Green, SC Barker. 2009. A Survey of Rhipicephalus microplus Populations for Mutations Associated With Pyrethroid Resistance. J Econ Entomol 102: 373-380.

[0017] Corley, SW, NN Jonsson, EK Piper, C Cutullè, MJ Stear, JM Seddon. 2013. Mutation in the RrrfiAOR gene is associated with amitraz resistance in the cattle tick Rhipicephalus microplus. PNAS 110: 16772-16777.

[0018] Corley, SW, EK Piper, NN Jonsson. 2012. Generation of full length cDNAs for eight putative GPCnR from the cattle tick, R. microplus using a targeted degenerate PCR and sequencing strategy. . Pios One 7: e32480.

[0019] Evans, PD, B Maqueira. 2005. Insect octopamine receptors: a new classification scheme based on studies of cloned Drosophila G-protein coupled receptors. Invertebrate Neuroscience 5: 111-118.

[0020] Farooqui, T. 2012. Review of octopamine in insect nervous systems. Insect Physiology 4: 1-17.

[0021] Faza, AP, IS Pinto, I Fonseca, GR Antunes, CM Monteiro, E Daemon, S Muniz Mde, MF Martins, J Furlong, MC Prata. 2013. A new approach to characterization of the resistance of populations of Rhipicephalus microplus (Acari: Ixodidae) to organophosphate and pyrethroid in the state of Minas Gerais, Brazil. Experimental Parasitology 134: 519-523.

[0022] Fernández-Salasa, A, Rl Rodriguez-Vivas, MA Alonso-Diaza. 2012. First report of a Rhipicephalus microplus tick population multi-resistant to acaricides and ivermectin in the Mexican tropics. Veterinary Parasitology 183: 338- 342.

[0023] Fernández-Salasa, A, Rl Rodriguez-Vivas, MA Alonso-Diaza, H Basurto-Camberosa. 2012. Ivermectin resistance status and factors associated in Rhipicephalus microplus (Acari: Ixodidae) populations from Veracruz, Mexico. Vet Parasitol.

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7/42 [0024] Fragoso-Sanchez, Η, Z Garcia-Vazquez, A Tapia-Perez, A OrtizNajera, R Rosario-Cruz, I Rodriguez-Vivas. 2011. Response of Mexican Rhipicephalus (Boophilus) microplus Ticks to Selection by Amitraz and Genetic Analysis of Attained Resistance. Journal of Entomology 8: 218-228.

[0025] Gibson, G, SP Muse. 2009. A primer of Genome Science. Sunderland, Massachusetts USA: Sinauer Associates, Inc. Pubishers.

[0026] Guerrero, FD, L Lovis, JR Martins. 2012. Acaricide resistance mechanisms in Rhipicephalus (Boophilus) microplus. Brazilian Journal of Veterinary Parasitology 21: 1-6.

[0027] Guerrero, FD, JH Pruett, AY Li. 2003. Molecular and biochemical diagnosis of esterase-mediated pyrethroid resistance in a Mexican strain of Boophilus microplus (Acari: Ixodidae). Exp Appl Acarol 28: 257-264.

[0028] Hall, T. 2007. BioEdit: Biological sequence alignment editor for Win95 / 98 / NT / 2K / XP [Online], [0029] Halliburton, R. 2003. Introduction to population genetics: Benjamin-Cummings Publishing Company.

[0030] Hartl, D, A Clarke. 1989. Principles of population genetics theory: Sinauer, Sunderland.

[0031] Hartl, DL. 2000. The Primer of Population Genetics Sunderland, MA. : Sinauer Associates ,.

[0032] He, H, AC Chen, RB Davey, GW Ivie, JE George. 1999. Identification of a Point Mutation in the para-Type Sodium Channel Gene from a Pyrethroid-Resistant Cattle Tick. Biochemical and Biophysical Research Communications 261: 558-561.

[0033] Hernandez, R, FD Guerrero, JE George, GG Wagner. 2002. Allele frequency and gene expression of a putative carboxylesterase-encoding gene in a pyrethroid resistant strain of the tick Boophilus microplus. Insect Biochemistry and Molecular Biology 32: 1009-1016.

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8/42 [0034] Holsinger, KE, BS Weir. 2009. Genetics in geographically structured populations: defining, estimating and interpreting FST. Nature Reviews, Genetics 10: 639-650.

[0035] Horak, IG, H Golezardy, AC Uys. 2007. Ticks associated with the three largest wild ruminant species in southern Africa. Onderstepoort J Vet Res 74: 231-242.

[0036] Jonsson, NN, C Cutullè, SW Corley, JM Seddon. 2010a. Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus microplus associated with resistance to flumethrin but not to cypermethrin. International Journal for Parasitology 40: 1659-1664.

[0037] Jonsson, NN, M Hope. 2007. Progress in the epidemiology and diagnosis of amitraz resistance in the cattle tick Boophilus microplus. Veterinary Parasitology 146: 193-198.

[0038] Jonsson, NN, RJ Miller, DH Kemp, A Knowles, AE Ardila, RG Verrail, JT Rothwell. 2010b. Rotation of treatments between spinosad and amitraz for the control of Rhipicephalus (Boophilus) microplus populations with amitraz resistance. Veterinary Parasitology 169: 157-164.

[0039] Katoh, Standley. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability, (outlines version 7). Molecular Biology and Evolution 30: 772-780.

[0040] Kliot, A, M Ghanim. 2012. Fitness costs associated with insecticide resistance. Pest Management Science 68: 1431-1437.

[0041] Lees, K, AS Bowman. 2007. Tick neurobiology: recent advances and the post-genomic era. Invertebrate Neuroscience 7: 183-198.

[0042] Lempereur, L, D Geysen, M Madder. 2010. Development and validation of a PCR-RFLP test to identify African Rhipicephalus (Boophilus) ticks. Acta Tropica 114: 55-58.

[0043] Li, AY, RB Davey, JE George. 2005. Carbaryl Resistance in Mexican Strains of the Southern Cattle Tick (Acari: Ixodidae). J Econ Entomol 98: 552-556.

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9/42 [0044] Li, AY, RB Davey, RJ Miller, JE George. 2004. Detection and Characterization of Amitraz Resistance in the Southern Cattle Tick, Boophilus microplus (Acari: Ixodidae). JOURNAL OF MEDICAL ENTOMOLOGY 41.

[0045] Li, AY, RB Davey, RJ Miller, JE George. 2005. Mode of inheritance of amitraz resistance in a Brazilian strain of the southern cattle tick, Boophilus microplus (Acari: Ixodidae). Experimental and Applied Acarology 37: 183-198.

[0046] Lyngso, RB, YS Song, J Hein. 2005. Minimum Recombination Histories by Branch and Bound.239-250.

[0047] M’diaye, K, M Bounias. 1991. Sublethal effects of the formamidine amitraz on honeybees gut lipids, following in vivo injections. Biomedical and Environmental Science 4: 376-383.

[0048] Madder, M, IG Horak. 2010. Tick Photodatabase. Tick Species. Belgium and Pretoria: Institute of Tropical Medicine Antwerpen-Belgium and the University of Pretoria, p. Rhipicephalus, Amblyomma, Hyalomma.

[0049] Maqueira, B, H Chatwin, PD Evans. 2005. Identification and characterization of a novel family of Drosophila β-adrenergic-like octopamine Gprotein coupled receptors. Journal of Neurochemistry 94: 547-560.

[0050] Morgan, JAT, SW Corley, LA Jackson, AE Lew-Tabor, PM Moolhuijzen, NN Jonsson. 2009. Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to synthetic pyrethroid acaricides. International Journal for Parasitology 39: 775-779.

[0051] Nachman, MW. 2006. Detecting selection at the molecular level. Evolutionary Genetics, Concepts and Case Studies.

[0052] Paris, M, F Roux, A Bérard, X Reboud. 2008. The effects of the genetic background on herbicide resistance fitness cost and its associated dominance in Arabidopsis thaliana. Heredity (Edinb) 101: 499-506.

[0053] Pohl, PC, GM Klafke, DD Carvalho, JR Martins, S Daffre, IdSV Jr., A Masuda. 2011. ABC transporter efflux pumps: A defense mechanism against

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10/42 ivermectin in Rhipicephalus (Boophilus) microplus. International Journal for Parasitology 41: 1323-1333.

[0054] Price, EW, I Carbone. 2005. SNAP: workbench management tool for evolutionary population genetic analysis. Bioinformatics 21: 402-404.

[0055] Robb, S, TR Cheek, FL Hannan, LM Hall, JM Midgley, PD Evans. 1994. Agonist-specific coupling of cloned Drosophila octopamine / tyramine receptor to multiple second messenger systems. EMBO Journal 16: 1325-1330.

[0056] Rosado-Aguilar, JA, RI Rodriguez-Vivas, Z Garcia-Vazquez, H Fragoso-Sanchez, A Ortiz-Najera, R Rosario-Cruz. 2008. Development of amitraz resistance in field populations of Boophilus microplus (Acari: Ixodidae) undergoing typical amitraz exposure in the Mexican tropics. Veterinary Parasitology 152: 349-353.

[0057] Saldivar, L, FD Guerrero, RJ Miller, KG Bendele, C Gondro, KA Brayton. 2008. Microarray analysis of acaricide-inducible gene expression in the southern cattle tick, Rhipicephalus (Boophilus) microplus. Insect Molecular Biology 17: 597-606.

[0058] Schnitzerling, HJ, J Nolan, S Hughes. 1983. Toxicology and metabolism of some synthetic pyrethroids in larvae of susceptible and resistant strains of the cattle tick Boophilus microplus (Can.). Pesticide Science 14: 64-72. [0059] Shin, DH, WH Hsu. 1994. Influence of the formamidine pesticide amitraz and its metabolites on porcine myometrial contractility: involvement of alpha 2-adrenoceptors and Ca2 + channels. Toxicology and Applied Pharmacology 128: 45-49.

[0060] Soberanes, N, MS Vargas, HF Sanchez, ZG Vazquez. 2002. First case reported of amitraz resistance in the cattle tick Boophilus microplus in Mexico. Tecnica Pecuaria en Mexico 40: 81-92.

[0061] Stone, BF, KP Haydock. 1962. A method for measuring the acaricide susceptibility of the cattle tick Boophilus microplus (Can.). Bulletin of Entomological Research: 563-578.

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11/42 [0062] Walker, AR, A Bouattour, JL Camicas, A Estrada-Pena, IG Horak, AA Latif, RG Pegram, PM Preston. 2003. Ticks of Domestic Animals in Africa: a Guide to Identification of Species. Edinburgh: Bioscience Reports.

[0063] Yeh, FC, RC Yang, T Boyle, ZH Ye, JX Mao. 1997. POPGENE: the user-friendly shareware for population genetic analysis. Molecular Biology and Biotechnology Center, University of Alberta, Canada.

Summary of the Invention [0064] Broadly in accordance with one aspect of the invention, a method is provided for detecting a genetic polymorphism associated with the susceptibility or resistance of an ectoparasite to an acaricide, the method comprising the step of screening a DNA sample from ectoparasite for the presence of one or more markers in the octopamine / tyramine receptor gene.

[0065] The method may include an additional step of determining ectoparasite homozygosity and heterozygosity.

[0066] The acaricide can be in the form of any of pyrethroids, formamidines or the like. The acaricide can be in the form of amitraz.

[0067] Ectoparasite can be in the form of a tick. The ectoparasite can be in the form of any of Rhipicephalus microplus, Rhipicephalus decoloratus or the like.

[0068] The one or more markers can be in the form of polymorphisms. The one or more markers can be in the form of single nucleotide polymorphisms (SNPs).

[0069] The markers can be in the form of either one or more of A22C and T65C, in which the presence of either or both of A22C and T65C is associated with ectoparasite resistance to the mite killer.

[0070] The one or more markers may include any one or more of C39T, G41A, G121A, T138C and C159T, in which their presence is associated with ectoparasite resistance to acaricide.

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12/42 [0071] The one or more markers can include either or both of T36C and G141C, in which the presence of T36C and / or G141C is associated with ectoparasite susceptibility to acaricide.

[0072] The determination of ectoparasite homozygosity and heterozygosity may include the classification of ectoparasites that have all markers associated with resistance, the markers being single nucleotide polymorphisms (SNPs) in the form of A22C, T65C, C39T, G41A, G121A, T138C and C159T, as homozygous resistant ectoparasites and ectoparasites that have only some of the markers associated with resistance, such as heterozygous ectoparasites.

[0073] The method can include any technique suitable for determining the presence or absence of one or more markers in the ectoparasite DNA sample, the technique includes any of: restriction fragment length polymorphism mapping, amplification reactions, hybridization from nucleic acids to specific allele probes or oligonucleotide arrays, various chip technologies, polynucleotide sequence techniques and combinations thereof.

[0074] The method can include the step of submitting the DNA sample for polynucleotide amplification using a pair of primers comprising SEQ. ID NO. 1 and SEQ. ID. AT THE. 2, and functional fragments, variants and mutations of each.

Primers for use in amplifying the octopamine / tyramine receptor SEQ.ID. NO.1 OAR-F172: 5'- agcattctgcggttttctac-3 ' SEQ.ID. AT THE. 2 OAR-R587: 5'- gcagatgaccagcacgttaccg- 3 '

[0075] Furthermore, according to the invention, a method of determining the susceptibility, or the reverse, of an ectoparasite to an acaricide, is provided, the method comprising the steps of:

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13/42 extraction of a DNA sample from ectoparasite;

amplification of a DNA sample sequence corresponding to at least part of an ectoparasite octopamine / tyramine receptor coding sequence;

obtaining an amplification product; and analysis of the amplification product for the presence of one or more markers associated with the susceptibility or resistance of ectoparasite to acaricide.

[0076] The method may include an additional step of determining the homozygosity and heterozygosity of ectoparasites.

[0077] The acaricide can be in the form of any of the pyrethroids, formamidines and the like. Specifically, the acaricide can be in the form of amitraz.

[0078] Ectoparasite can be in the form of a tick. Specifically, ectoparasite can be in the form of any one of Rhipicephalus microplus, Rhipicephalus decoloratus and the like.

[0079] The one or more markers can be single nucleotide polymorphisms (SNPs). SNPs can be any one or more of A22C and T65C in which the presence of either or both of A22C and T65C is associated with ectoparasite resistance to acaricide. The one or more SNPs can include any one or more of C39T, G41A, G121A, T138C and C159T in which their presence is associated with ectoparasite resistance to acaricide. The one or more SNPs can include T36C and G141C in which the presence of T36C and / or G141C is associated with ectoparasite susceptibility to acaricide.

[0080] The determination of ectoparasite homozygosity and heterozygosity may include the classification of ectoparasites having all SNPs associated with resistance (A22C, T65C, C39T, G41A, G121A, T138C and C159T) as homozygous and ectoparasite resistant ectoparasites having only a few

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14/42 of the SNPs associated with resistance (not all), such as heterozygous ectoparasites.

[0081] The DNA sample can be amplified using a primer pair selected from the SEQ nucleotide sequences. ID NO. 1, SEQ. ID. AT THE. 2, and functional fragments, variants and mutations of each.

[0082] The step of analyzing the amplification product for the presence of one or more markers associated with ectoparasite resistance to acaricide can be by way of sequencing the amplification product, quantifying the amplification product, detecting a probe attached to the product amplification, and use of a primer extension reaction (PEXT) visualized through an immersion test or similar.

[0083] The step of analysis of the amplification product may include exposure of the amplification product for digestion of the restriction enzyme.

[0084] Typically, restriction digestion can be performed by using a restriction enzyme having a recognition site corresponding to at least part of one or more markers. In one embodiment, restriction digestion can be performed by a restriction enzyme having a recognition sequence comprising 5'-GGCGGA-3 '(SEQ. ID. NO. 3). The restriction enzyme can be Ec / 1.

Alternatively or additionally, restriction can be accomplished by using a restriction enzyme having a recognition sequence comprising 5'-GGACG -3 '(SEQ. ID. NO. 4). Consequently, the restriction enzyme can be selected from the group consisting of any one or more of: BseG \, BstF5 \, BsíPZ418l, Fok \, Sfôl and any enzyme that recognizes a site corresponding to at least part of the one or more markers.

[0086] After digestion, the resulting fragments can be separated at least partially from each other using size separation techniques, such as gel electrophoresis and the like.

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The markers can be DNA-based markers selected from the group consisting of A22C and T65C of the coding sequence of the amplified DNA sequence using the method of the invention.

[0088] The markers can be peptide-based markers selected from the group consisting of G14E, T8P and L22S.

[0089] The invention extends to the use of any one or more of the genetic markers C39T, G41A, G121A, T138C and C159T as indicators of ectoparasite resistance to acaricide. The invention also extends to the use of any one or more of the genetic markers T36C and G141C as indicators of ectoparasite susceptibility to acaricide.

[0090] The stage of extracting a DNA sample from the ectoparasite can be in the form of extracting DNA from any of the larvae or whole ticks.

[0091] In an embodiment in which DNA is extracted from whole adult ticks, a modified salt-based extraction method can be used to isolate genomic DNA.

[0092] The modified salt-based extraction method includes the steps of:

provision of whole tick samples;

homogenization of whole ticks in 200 μΙ_ of lysis buffer (0.5 M EDTA, 0.5% (w / v) sodium lauroyl sarcosinate).

addition of an additional 400 μΙ_ of DNA extraction solution (0.4 M NaCI, 60 mM Tris-HCI, 12 mM EDTA, 0.25% SDS, pH 8.0) for samples with 2 μΙ_ of proteinase K (15 mg / ml), well mixed and incubated overnight at 55 ° C;

incubation of the samples for 20 min at 65 ° C to inactivate proteinase K, after which 1 μΙ_ of RNase A (10 mg / mL) is added;

briefly vortexing the samples;

incubation of samples at 37 ° C for 15 min;

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16/42 carrying out protein precipitation by adding 360 μΙ_ of 5 M NaCI, vortexing for 10 seconds, and incubating on ice for 5 minutes, followed by centrifugation at 25500 xg for 20 minutes at room temperature, to provide a supernatant;

adding an equal volume of isopropanol to the supernatant, vortexing briefly, followed by a 1 hour incubation at -20 ° C;

centrifugation of the samples for 20 minutes at 10,000 xg and disposal of supernatants; washing DNA pellets three times in a row with 500 μΙ_ of 70% ethanol, centrifuged for 5 minutes at 10,000 xg, and discarding the supernatant; and air drying the final DNA pellets; and resuspension of 50 μΙ_ air-dried DNA pellets in 1 x TE buffer (1 mM Tris-HCI, 0.1 mM EDTA, pH 7.0).

[0093] In an embodiment in which Genomic DNA is extracted from individual larvae, the extraction method includes crushing the individual larvae in a 1.5 ml microcentrifuge tube containing 25 μΙ_ of TE buffer (10 mM Tris, EDTA at 1 mM, pH 7.6) to form a suspension;

boiling the suspension for 5 minutes;

centrifugation at 4000 xg for 30 seconds at room temperature to provide a supernatant; and using the supernatant for PCR.

[0094] The DNA sample amplification step can be performed by submitting the DNA sample to the following heating, annealing and cooling conditions, respectively:

heat to 94 ° C for 4 minutes;

followed by 40 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 1 minute; and with a final extension of 72 ° C for 8 minutes.

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17/42 [0095] The invention also extends to an isolated nucleic acid molecule selected from the group consisting of:

(i) a nucleic acid molecule comprising the SEQ sequence. ID. AT THE. 1;

(ii) a nucleic acid fragment having a sequence derived from an octopamine / tyramine receptor gene and containing any one or more of the markers associated with ectoparasite resistance to an acaricide, the markers selected from: A22C, T65C, C39T, G41A , G121A, T138C and C159T;

(iii) a nucleic acid fragment having a sequence derived from an octopamine / tyramine receptor gene and containing any or more of the markers associated with an ectoparasite susceptibility to an acaricide, the markers selected from: T36C and G141C; and (iv) sequences complementary to that.

[0096] The isolated nucleic acid molecule may be in the form of an unnaturally occurring sequence.

[0097] The unnaturally occurring sequence may be in the form of cDNA.

[0098] The invention further provides an amplified polynucleotide having a polymorphism in the form of any one or more of: A22C, T65C, C39T, G41A, G121A, T138C, C159T, T36C and G141C.

[0099] The invention also extends to the use of an isolated nucleic acid molecule as described or an amplified polynucleotide as described in a method of detecting a genetic polymorphism associated with susceptibility or resistance of an ectoparasite to an acaricide.

[0100] The invention further provides for the use of any one or more of the genetic markers C39T, G41A, G121A, T138C and C159T as indicators of ectoparasite resistance to an acaricide.

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18/42 [0101] The invention extends to the use of either or both of the genetic markers T36C and G141C as indicators of the susceptibility of an ectoparasite to an acaricide.

[0102] The invention also extends to use any one or more of the genetic markers in the form of peptides based on G14E, T8P and L22S as indicators of ectoparasite resistance to an acaricide.

[0103] The invention also provides a kit to determine the susceptibility, or the reverse, of an ectoparasite or a group of parasites to an acaricide, the kit including:

primers in the form of SEQ. ID. NO.1 and SEQ. ID. AT THE. 2; and a suitable reagent buffer to allow amplification of a DNA sequence to be amplified using the primers.

[0104] The kit may include instructions for determining the susceptibility, or the reverse, of an ectoparasite to an acaricide.

[0105] All references to tick DNA sequences and single nucleotide polymorphisms (SNPs) refer only to nucleotide sequences in the coding region; positions of non-coding regions are excluded.

[0106] Additional aspects of the invention will now be described by way of non-limiting examples with reference only to the accompanying drawings.

Drawings [0107] Figure 1 shows sequence alignment of a portion of the OCT / Tyr / tyramine receptor gene for R. microplus larvae resistant and susceptible to amitraz. Rhipicephalus (Boophilus) microplus was the reference sequence used for the alignments (Access to GenBank: AJ010743.1) with the resistant strain Santa Luiza (Access to GenBank: EF490688.1) and the susceptible strain Gonzalez (Access to GenBank: EF490687. 1). The five samples aligned below the Santa Luiza strain are the resistant samples, and those

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19/42 below the Gonzalez strain are those susceptible. The gray blocks indicate two SNPs associated with the resistance, namely, A22C and T65C.

[0108] Figure 2 shows an amino acid sequence of a portion of the OCT / Tyr / tyramine receptor gene. The non-coding region of the gene is indicated from the position of the amino acid from 1 to 45. Seven substitutions occur within the non-coding regions and six in the open reading frame. The gray blocks highlight the two substitutions associated with resistance.

[0109] Figure 3 shows the distribution of mean diversity versus the number of loci for the OCT / Tyr receptor. A comparison of 22 different loci within the OCT / Tyr / tyramine receptor is shown. Plateau diversity implies that there are sufficient samples for further analysis.

[0110] Figure 4 shows the ^rd density distribution for the OCT / Tyr / tyramine receptor. The ^rd values are placed on the x axis while the relative occurrence of each of these values is displayed on the y axis. The observed value is outside the distribution range generated by the randomized data set.

[0111] Figure 5 shows the ancestral recombination chart for homozygous R. microplus ticks resistant and susceptible to amitraz in South Africa. H1, H2, H3, H4 and H5 represent the sequences of haplotypes compatible with infinite locations that have been observed. The E node represents the ancestral form of the gene with adjacent points (A-D) illustrating coalescent haplotypes. The additional table indicates the sample / haplotype associations for the graph. A recombination event was detected as the predecessor of the H2 haplotype, and 294 indicating that the recombination event occurred between nucleotides 294 and 295 in the alignment. The symbol S means that the suffix for the recombinant was provided by the predecessor of the recombinant and H3, while P means that the prefix (the first 294 nucleotides) was provided by the predecessor of the recombinant and H5. South Africa samples are

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20/42 labeled with a number indicating the holding of origin and MF indicates R. microplus females.

[0112] Figure 6 shows an electropherogram of agarose gel of the OCT / Tyr / tyramine receptor of R. decoloratus larvae digested with Ec / 1 restriction enzyme. The track numbers represent samples from the original set of 14 anonymous samples. Range 1-sample 5, range 2-sample 7, range 3-sample 8, range 4-sample 6 and range 5-sample 9 (Supplementary Table S3).

[0113] Figure S1 shows the subpopulation structure of ticks across South Africa.

Detailed Description of the Invention [0114] All references to tick DNA sequences and single nucleotide polymorphisms (SNPs) refer to the nucleotide sequences of the coding region only; positions of non-coding regions are excluded.

1. Results

Screening for SNPs in resistant larvae and susceptible to amitraz [0115] The larvae of R. microplus resistant to amitraz obtained from the Mnisi area in Kruger National Park were screened for the presence of the two resistant SNPs published by Chen et al. (2007). Twenty-four nucleotide substitutions were detected among the resistant larvae, susceptible larvae and the three NCBI reference strains (Figure 1). Seven of the substitutions appeared to be associated with susceptible samples. The first four of these substitutions occurred in the non-coding region (position of nucleotide 1-135) of the gene consisting of a transverse and three transition mutations. The three remaining substitutions within the open reading frame were synonymous, having no observable effect on the amino acid sequence. There were four substitutions in all resistant samples (Figure 1) and contained two non-synonymous locations at nucleotide positions 157 and 200 (Figure 2, T8P and L22S). These two substitutions correspond to two published SNPs. There are seven nucleotide substitutions differentiating the

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21/42 Boophilus microplus protein coupled receptor (GenBank Access AJ010743.1) from the other sequences, three of which occur in the coding region and two non-synonymous changes in the coding region (115V and T20A) (Figure 2). Finally, there are several other SNPs that appear to appear with two non-synonymous substitutions that occur at nucleotide positions 176 and 256 (Figure 1). Samples containing these additional SNPs were resistant.

[0116] The sequence data obtained for all larvae were compared to the results of the LPTs (Supplementary Table S1). The comparison reveals a clear correlation between the presence of the SNP (genotype) and the resistant phenotype determined by the LPTs. The comparison was made primarily between the larvae that survived the LPT test (resistant) and those that did not survive (susceptible). The results clearly show that all larvae that did not survive the LPT test exhibit the SS or RS genotype, and those that survived amitraz exposure exhibit the RR genotype.

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Table S1. Correlation between state of resistance to phenotypic amitraz (LPT) and genotypic (sequencing)

Sample Larvae that survived LPT ^to % of Amitraz Strain strain ^c Genotype ⁰ Phenotype 5AM2 (4) A2 0.4 100 RR Amitraz resistant 5AM2 (4) A3 0.4 100 RR Amitraz resistant 5AM3 (8) A1 0.08 100 RR Amitraz resistant 5AM3 (8) A3 0.08 100 RR Amitraz resistant 3AM2 (4) A3 0.4 100 RR Amitraz resistant 2AM3 (16) A3 0.016 28 RR Amitraz resistant 1AM3 (8) A3 0.08 10 RR Amitraz resistant

Larvae that did not survive LPTs ^b

Sample Amitraz RF% of strain ^c Genotype ^{0 1} Phenotype 2AM3 (16) A1 0.016 28 SS Susceptible to Amitraz 2AM3 (16) A2 0.016 28 SS Susceptible to Amitraz 1AM3 (8) A2 0.08 10 SS Susceptible to Amitraz 1AM3 (8) A3 0.08 10 SS Susceptible to Amitraz 1AM (8) 1 0.08 10 LOL Susceptible to Amitraz 1AM3 (16) 1 0.016 10 LOL Susceptible to Amitraz ^the Larvae that They were alive after completing the rehearsal of larval pack testing,

meaning that they were resistant to the concentrations of amitraz applied. ^b Larvae that were susceptible to the concentrations of amitraz used and were killed after completing the test. ^C RF is the resistance factor, and RF 100 implies that the strain is resistant to amitraz. RF of 10 and 28 means that the strain is susceptible to amitraz. ^d Genotype was inferred based on whether or not the sequenced larvae contained the two SNPs published by Chen et al. (2007). RR - resistant to homozygous, SS susceptible to homozygous, RS - heterozygous.

National screening for SNPs in adult ticks [0117] Adult ticks from 108 different farms showed a 50% incidence of R. microplus. Most of the ticks collected were from Kwazulu-Natal Province. Therefore, a total of 218 alleles

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23/42 (109 adult ticks from all over South Africa) was tracked for the two published SNPs.

[0118] Several additional SNPs were found within field samples across South Africa, mainly within the non-coding region of the OCT / Tyr receptor gene (data not shown). The frequencies of susceptible (S) and resistance (R) alleles were calculated for the SNPs linked to the resistance that occurs in the OCT / Tyr receptor gene for all adult ticks. Almost half of the population (48.2%) exhibited resistance alleles for amitraz, and the majority of the population was heterozygous in these nucleotide positions (Supplementary Table S2).

Table S2. Allele frequencies for the octopamine receptor gene in R. microplus field samples.

Sample Alleles Sample Alleles Sample Alleles 44.2MF SS 45.1 MF SS 70.2MF LOL 46.2MF LOL 45.4MF SS 7.5MF LOL 46.3MF LOL 45.5MF RR 7.6MF SS 46.5MF LOL 21.1MF SS 37.2MF LOL 44.3MF SS 50.3MF SS 41.2MF LOL 44.5MF LOL 66.1 MF SS 41.11MF LOL 44.4MF LOL 66.2MF SS 41.12MF LOL 71.1MF LOL 66.3MF LOL 41.13MF LOL 17.1MF RR 66.5MF RR 49.1 MF SS 20.1 MF SS 66.6MF LOL 49.6MF LOL 20.2MF SS 67.1 MF LOL 49.8MF RR 20.3MF SS 67.2MF RR 49.10MF LOL 26.7MF LOL 67.3MF SS 58.1 MF LOL 26.8MF LOL 67.4MF LOL 58.3MF LOL 26.1 MF LOL 67.5MF RR 58.7MF RR 26.6MF RR 67.7MF SS 58.11MF RR 47.2MF LOL 51.1MF LOL 58.13MF LOL 54.1 MF RR 51.2MF RR 93.1 MF LOL 54.7MF LOL 51.3MF LOL 93.2MF LOL 54.9MF LOL 51.4MF LOL 93.3MF LOL 54.10MF RR 51.5MF RR 93.4MF LOL 73.1 MF RR 69.3MF LOL 93.5MF LOL 73.2MF LOL 69.4MF LOL 93.7MF RR

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73.3MF LOL 69.5MF LOL 93.8MF LOL 73.9MF LOL 9.2MF RR 95.1 MF LOL 73.4MF LOL 9.1 MF RR 95.3MF LOL 73.5MF LOL 9.3MF LOL 95.4MF LOL 73.7MF LOL 77.1 MF LOL 95.6MF LOL 73.8MF SS 9.6MF RR 37.4MF LOL 86.1 MF SS 62.1 MF SS 40.3MM SS 86.2MF SS 62.2MF SS 42.1 MF LOL 86.3MF LOL 62.3MF SS 18.2MF SS 86.4MF RR 62.4MF SS 18.1MF LOL 86.5MF LOL 62.5MF LOL 86.6MF LOL 65.1 MF RR 86.7MF SS 65.2MF RR 86.8MF LOL 65.3MF SS Allele R 0.482 70.1 MF LOL 79.1 MF LOL Allele S 0.518

0 ¹ R allele refers to the mutant form which gives rise to resistance as the S allele refers to susceptible wild - type form of the gene.

Qametic imbalance between SNPs [0119] In this study, all SNPs that were present at the OCT / Tyr receptor were analyzed to determine whether they were in gametic imbalance. First, diversity was modeled against the number of loci (variable positions in the sequences) (Figure 3). This is essential to determine whether there are sufficient samples for the proposed analysis. The graph of average diversity versus number of loci should reach a plateau, meaning that there are sufficient samples for further analysis (which is evident in Figure 3). Thus, the additional samples or loci would not have altered the diversity that was observed in the sample set.

[0120] An analysis of gametic imbalance revealed a graph with a bell-shaped curve, with the observed value ^rd being outside the general distribution (Figure 4). This implies that the observed ^rd value was significantly higher (P <0.00001; H _o = random association or gametic imbalance) than was generated by the randomized data set, thus emphasizing a deviation from the random association. Therefore, the data

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25/42 observed exhibited a significant level of gametic imbalance (non-random association), while gametic balance (random association) was absent.

[0121] Initial comparisons were made for the two SNP loci that were shown to be involved in amitraz resistance (loci 11 and 17). Locus 11 corresponds to the position of nucleotide 157, and locus 17 to that of 200. A paired comparison of these two loci resulted in an ^rd value of 0.804 (P <0.00001), indicating that these two sites are in gametic imbalance. The homozygous resistant samples showed that both substitutions were present. Paired comparisons were then made for all variable loci within the gene (data not shown). Table 1 only shows the results of the loci that were significantly associated with the two resistant loci.

Table 1. Loci significantly associated with resistant loci 11 and 17.

SNP 1 SNP 2 value ^a Aggregate ^b Value of P

Locus 11 Locus 12 0.834 <0.00001 Locus 17 Locus 12 0.921 0.877 <0.00001 Locus 11 Locus 13 0.872 <0.00001 Locus 17 Locus 13 0.884 0.878 <0.00001 Locus 17 Locus 19 0.803 0.803 <0.00001

^{a rd} value represents the gametic imbalance that is observed between the two SNPs being compared to each other.

^b Added value is the average value of the ^rd values that were obtained for loci 11 and 17 associated with the other loci to determine which association exhibited the greatest gametic imbalance.

[0122] Locus 12 corresponds to nucleotide position 171 in the alignment of the OCT / Tyr receptor (Figure 1), locus 13 to that of position 174, and locus 19 to that of 273. The aggregated ^rd value was calculated to determine which of the three loci were more strongly associated with the two loci

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26/42 resistant (11 and 17). The results show that the resistant loci are equally associated with both 12 and 13. When comparing the sequence data, it is evident that a nucleotide substitution at locus 12 only occurs when the tick is suspected of being susceptible to amitraz. A replacement at locus 13, however, occurs every time the tick is suspected of being resistant. Therefore, it may be possible to consider this particular association as a molecular marker for resistance in the gene.

[0123] The population structure was also analyzed by Wright's Fst differentiation measure for the data set, and generated a value of 0.0253 (Holsinger, Weir 2009). This value implies that the population subdivision represents 2.5% of the total genetic variation seen within the total population. Additionally, the observed (H _o ) and expected (H _e ) heterozygosis for each subpopulation was calculated, with the inbreeding coefficient (Fis) (Table 2). All Fis values were negative, with the exception of population 3. The global analysis in all populations was also performed and generated a global inbreeding coefficient of -0.137, indicating excess heterozygosity compared to what was expected.

Table 2. Genetic variation among tick populations in South Africa

Population n Ho H _and Fis HWE 1 8 0.7500 0.5000 -0.6000 2 31 0.5161 0.5034 -0.0420 3 21 0.4762 0.5110 0.0455 4 5 0.4000 0.3556 -0.2500 5 39 0.6410 0.5052 -0.2854 General 104 0.5670 0.49982 -0.1370 Excess of heterozygotes

Observed heterozygosis (H _o ), expected (H _e ) and fixation indices (F, _s ) for each subpopulation.

Recombination in the OCT / Tyr receptor gene

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27/42 [0124] The ancestral recombination graphs were constructed by incorporating sequence information from the OCT / Tyr receptor gene of both resistant and susceptible homozygous ticks (Figure 5).

[0125] A recombination event occurred at nucleotide position 294. There were no mutations between the recombination event and the H2 haplotype, so H2 is the recombinant. When looking at the sequence data, the nucleotide substitution that occurs at that location is C to T. This substitution is synonymous and only appears in samples suspected of being resistant. This replacement occurred at a frequency of 0.61 in the R. microplus tick population in South Africa (data not shown), especially within heterozygous samples where both susceptible and resistant alleles were present. No geographical significance was found with the haplotype sample clusters and the area in South Africa where the samples originated.

Diagnostic tool based on RFLP for resistance to amitraz:

[0126] Locus 13, corresponding to nucleotide position 174, was significantly associated with the two resistant loci in the OCT / Tyr receptor gene (Table 1). A nucleotide substitution from a C to a T was present whenever the tick was resistant in a phenotypic and genotypic way, that is, both SN Ps associated with the resistance were present. This was the case for both homozygous and heterozygous SNP alleles. However, this substitution did not result in an amino acid change, due to the degeneration of the third position of the codon. Such a tight association between loci in the gene could potentially represent a molecular marker for resistance to amitraz.

[0127] R. decoloratus ticks with known resistance and susceptibility to amitraz were used to test the effectiveness of the Ec / Ί restriction enzyme as a possible diagnostic tool. Fourteen samples

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Anonymous 28/42 were obtained, each containing multiple larvae. Of these, five samples were used for RFLP analysis (Figure 6).

[0128] Sample 5 (Range 1, Figure 6) showed a prominent band at 400 bp, indicating a homozygous genotype and resistant phenotype. This result was corroborated by an independently carried out larval pack test (Supplementary Table S3). This particular sample exhibited 13.2% control at a field concentration of 250 ppm amitraz. Samples 7 and 8 (Lanes 2 and 3 respectively, Figure 6) produced identical digestion products. In addition to the 400 bp digest product, a 223 bp and 186 bp product was also detected. This result means that these samples were heterozygous and were potentially more susceptible to treatment with amitraz. In fact, independent larval package tests revealed that these samples were 100% susceptible to amitraz field concentrations. Sample 6 (Range 4, Figure 6) showed resistance potential in the same way as Sample 5, and this was corroborated by a 30% level of control during the independent larval packet tests (Supplementary Table S3). Finally, Sample 9 (lane 5, Figure 6) showed heterozygosity in the same way as in Samples 7 and 8, and was 100% susceptible to amitraz field concentrations (Supplementary Table S3).

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Table S3. Rhipicephalus microplus larval pack test results

Sample number Collection date Tick species % control in field concentration 1 03 March 09 R. dec 0 2 ?? April 09 R. dec 100 3 02 February 10 R. dec 100 4 18 March 10 R. dec 11.8 5 06 May 11 R. dec 13.2 6 16 February 11 R. dec 30 7 26 May 10 R. dec 100 8 19 February 13 R. dec 100 9 21 February 13 R. dec 100 10 30 January 09 R. dec 11.8 11 29 April 09 R. dec 100 12 25 May 10 R. dec 100 13 13 April 12 R. dec 10.9 14 20 February 09 R. dec 34.7

² 90 to 100% - Amitraz considered to be effective, 80 to 90% - effective with reserve, 50 to 80% - indications of resistance in development, 0 to 50% - indications of resistance. Amitraz concentration was 250 ppm.

2. Discussion [0129] In this study, the evolution of resistance to amitraz in South African Rhipicephalus microplus ticks was investigated. Two SNP loci previously known at the OCT / Tyr receptor (Chen et al., 2007) were significantly associated with each other, and with resistance to amitraz. Finally, the high level of gametic imbalance between these two SNPs and an additional SNP, which changes a restriction enzyme binding site, was explored in order to invent an accessible and easy restriction enzyme based on the resistance assessment tool amitraz in tick field samples.

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30/42 [0130] As previously mentioned, octopaminergic receptors have been classified into these three classes. Α-adrenergic octopamine receptors exhibit an increased affinity for octopamine, rather than tyramine, which leads to an increase in intracellular Ca ²⁺ concentrations with a small increase in intracellular cAMP levels (Balfanz et al. 2005; Farooqui 2012). Βadrenergic type octopamine receptors, on the other hand, are specifically activated in response to octopamine directly resulting in increased levels of intracellular cAMP (Maqueira, Chatwin, Evans 2005; Farooqui 2012). Finally, octopaminergic / tyramergic receptors (OCT / Tyr) showed immense similarity in terms of structure and pharmacology with vertebrate α2adrenergic receptors (Evans, Maqueira 2005). Agonistic preferences can result in receptors that are stimulated by octopamine or tyramine. In response to octopamine, there will be an increase in intracellular Ca ²⁺ concentrations. Conversely, a response to tyramine will result in a reduction in intracellular cAMP levels (Farooqui 2012). Robb et al. (1994) also demonstrated this principle in Drosophila, where a recipient can display different pharmacological profiles with respect to implemented second messenger systems.

[0131] Previous studies on bees (M'diave, Bounias 1991) and mammals (Shin, Hsu 1994) suggested that the proposed target site for amitraz was α-adrenergic receptors and a2adrenergic receptors, respectively. This, together with the SN Ps discovered by Chen, He, Davev (2007), strongly suggests the involvement of OCT / Tyr type receptors in amitraz resistance. The mutation reported by Corley et al. (2013) in the β-adrenergic type octopamine receptor seems promising; however, its location restricted only to central Queensland in Australia further substantiated the notion to investigate the OCT / Tyr receptor as an alternative.

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31/42 [0132] A national screening of the two SNPs associated with resistance in the OCT / Tyr receptor gene revealed a high level of heterozygosity. It is assumed that the selection of positive equilibrium is acting in the population in order to maintain the high prevalence of heterozygosis (Nachman 2006). The selection pressure is imposed on the tick population by the application of amitraz, and this triggers alleles that confer resistance to homozygosis (Soberanes et al. 2002; Rosado-Aquilar et al. 2008). On the other hand, the potential advantages of heterozygosis without amitraz selection pressure can make subsections of the population that escape amitraz treatment remain heterozygous. This observation could be further explained by a slow rate of fixation during constant selective pressure, such as in tick populations that were under pyrethroid selection pressure (Faza et al. 2013). Studies have also shown that there is a lack of fitness with strains resistant to amitraz (Jonsson et al. 2010b), thus providing insight into the selective disadvantage that can be associated with homozygous resistant ticks. This particular fitness cost would largely contribute to the excess heterozygotes found within the study. Such interactions between selection pressure and fitness cost will determine the frequency of allele associated with resistance and can occur through direct or pleiotropic effects (Kliot, Ghanim 2012).

[0133] The two SNP loci associated with putative resistance were in gametic imbalance and were positively associated with the resistant phenotype. Thus, if both SNP loci exhibit specific nucleotide substitutions, it can be inferred that the tick is resistant to amitraz. Although there is a significant gametic imbalance for these SNPs, there is a small but important deviation from the complete imbalance. The gametic imbalance between loci can be generated by a variety of factors, namely: mutation, population mix, gene flow, genetic drift, some types of natural selection, as well as genetic heterogeneity (Halliburton 2003; Gibson, Muse 2009). The coalescing interaction of these forces within the

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32/42 tick population is probably responsible for the incomplete imbalance observed.

[0134] A third SNP position in the OCT / Tyr receptor gene was significantly associated with the two SNPs previously mentioned. This illustrates that resistance to amitraz is potentially associated with complete alleles of the gene, rather than with individual SNP positions. Therefore, associations between SNP positions could potentially be attributed to intact allele inheritance. This is further demonstrated by additional variable positions that seemed to distinguish resistant from susceptible phenotypes. Through constant selection pressure, these variable positions reached the population. Therefore, it appears that exposure to amitraz consequently affects a wider range of loci, further substantiating the probability of intact allele inheritance. A classic evolutionary process that could be responsible for such an observation is epistatic interaction, where these combination alleles within the gene could offset the fitness cost mentioned earlier (Paris et al. 2008).

[0135] It was investigated whether intragenic recombination could be inferred from a population sample of the OCT / Tyr receptor gene. In fact, the recombination has been detected, and is unlikely to be an analysis artifact, since the inferred recombination site is 294 bp in the sequenced section of the gene. The recombination in the OCT / Tyr receptor gene would allow new combinations of the prefix and suffix of the gene, which would allow new combinations of SNP, as well as the appearance of double homozygotes in the population. Our results also showed that such double homozygotes exhibited a very high level of resistance to amitraz. Therefore, it is likely that under constant pressure of amitraz selection, recombination in the OCT / Tyr receptor gene could play an important role in the emergence of resistance to this acaricide. In addition, recombination can act to generate or decay allelic combinations (Halliburton 2003); with natural selection acting on these combinations of alleles that they can be kept in

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33/42 population (Gibson, Muse 2009). This explains the inheritance of complete allele observed within this population. The relative strength of these two processes will determine the strength of the imbalance between the loci (Halliburton 2003).

[0136] Gametic imbalance will be transitory in the absence of amitraz selection pressure acting to maintain it, thus facilitating the recombination that will break non-random allelic associations (Hartl, Clarke 1989; Halliburton 2003). This could explain the additional variable sites that were detected in heterozygotes that exhibited minimal associations with other loci (data not shown). As previously mentioned, the heterozygous subsections of the population could escape the pressure of amitraz, thus preventing the imbalance and assisting the recombination that gives rise to these new combinations of alleles.

[0137] Population differentiation studies showed that the inbreeding coefficient (Fis) within subpopulations was negative for all but one population, meaning that most individuals are heterozygous. The global inbreeding coefficient was -0.137, and indicated an excess of heterozygotes over all analyzed populations (Holsinger, Weir 2009). This could be due to a number of factors including dissociated mating, breach of isolation or a Wahlund effect where excess heterozygotes are observed on the expectation of HWE (Hartl 2000). The general degree of genetic differentiation (Fst) revealed that 2.5% of the genetic variation was distributed among the subpopulations, while the remaining 97.5% was due to the variation within the subpopulations.

[0138] The exploration of the existence of associated SN Ps resulted in the accurate detection of resistance to amitraz in synonymous samples. Molecular detection using an RFLP technique has been shown to be cheaper, easier and faster than traditional larval packet testing. Heterozygotes have been characterized as susceptible, substantiating previous claims that resistance to amitraz is necessarily inherited (Fragoso-Sanchez et al. 2011). Most of the

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34/42 population exhibited heterozygosity, through extrapolation of the results obtained from RFLP evaluations, it can be suggested that this population is still susceptible to amitraz (with the exception of resistant samples of homozygotes). This protocol will play an important future role in monitoring the emergence of resistance to amitraz in South African R. microplus and R. decoloratus ticks. Such information could assist in guiding the use of effective control chemicals, as well as predicting when and where resistance to amitraz may appear.

[0139] The novelty of this work includes the first confirmation of the two SNPs published by Chen et al. (2007) in larvae resistant to amitraz, as well as field populations of ticks. It was also the first report of any association studies conducted for the OCT / Tyr receptor in ticks. The discovery of a recombination event that could possibly be the 'switcher' of resistant and susceptible phenotypes in populations exposed to amitraz is a significant advance towards a more complete perspective on the evolution of resistance to acaricide. Finally, this study is the first indication that heterozygous field populations are susceptible to treatment with amitraz, allowing for alternative and improved strategies to be implemented on farms before the total developed resistance is acquired.

3. Materials and Methods

Collections, identification and extraction of tick DNA [0140] Adult ticks were collected by Zoetis (Pty) Ltd. from 108 different farms across South Africa, of which 53 farms contained R. microplus ticks. The classification of ticks collected in their respective genera was made according to previous authors (Walker et al. 2003; Madder, Horak 2010). Another differentiation between species of Rhipicephalus (Boophilus) was made using microscopy techniques. To distinguish between R. microplus and R. decoloratus females, the hypostoma dentition was examined, with adanal spurs for male ticks (Walker et al.

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2003; Madder, Horak 2010). The molecular identification of ticks was also performed based on the analysis of restriction fragment length polymorphism (RFLP) (Lempereur, Gevsen, Madder 2010).

[0141] Control samples of larvae resistant to amitraz were obtained from the Mnisi area in Dr. Rosalind Malan's Kruger National Park. Twelve different strains were provided, of which three were classified as resistant based on their resistance factors, which were determined by conventional larval pack tests (Stone, Havdock 1962).

[0142] A modified salt-based extraction method published by Aljanabi, Martinez (1997) was used to isolate genomic DNA from whole adult ticks (predominantly female ticks). The whole ticks were homogenized in 200 μΙ_ of lysis buffer (0.5 M, 0.5% (w / v) sodium lauroyl sarcosinate). An additional 400 μΙ_ of DNA extraction solution (0.4 M NaCl, 60 mM Tris-HCl, 12 mM EDTA, 0.25% SDS, pH 8.0) was added to the samples with 2 μΙ_ of proteinase K (15 mg / mL), well mixed and incubated overnight at 55 ° C. The samples were incubated for 20 minutes at 65 ° C to inactivate proteinase K, after which 1 μΙ_ of RNase A (10 mg / mL) was added. The samples were briefly vortexed and further incubated at 37 ° C for 15 minutes. Protein precipitation was performed by adding 360 μΙ_ of 5 M NaCl, vortexing for 10 seconds, and incubating on ice for 5 minutes, followed by centrifugation at 25500 xg for 20 minutes at room temperature. An equal volume of isopropanol was added to the supernatant, briefly vortexed, followed by a 1 hour incubation at -20 ° C. The samples were then centrifuged for 20 minutes at 10,000 xg and the supernatants discarded. The DNA pellets were washed three times in a row with 500 μΙ_ of 70% ethanol, centrifuged for 5 minutes at 10,000 xg, and the supernatant discarded. The final DNA pellets were dried with air and then resuspended in 50 μΙ of 1 x TE buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 7.0).

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36/42 [0143] Genomic DNA was extracted from individual larvae using a modification of the protocol by Hernandez et al. (2002). Briefly, the individual larvae were crushed in a 1.5 ml microcentrifuge tube containing 25 μΙ of TE buffer (10 mM Tris, 1 mM EDTA, pH 7.6). The suspension was then boiled for 5 min and centrifuged at 4000 xg for 30 seconds at room temperature. The supernatant was used directly for PCR.

PCR amplification and OCT / Tyr receptor gene sequencing [0144] Published primers (Chen et al. 2007) were used for PCR amplification of a 417 bp fragment of the OCT / Tyr receptor gene at an annealing temperature of 55 ° Ç. This method worked sufficiently for some samples, but due to differences in the tick strain, a new direct primer was designed. The new direct primer, OARF172 (5'- AGC ATT CTG CGG TTT TCT AC-3 '), was designed based on the sequence data from the new samples that have successfully amplified. This direct primer was used for most tick samples with the same reaction conditions. The use was made of the following reverse primer: OAR-R587 (5 '- GCA GAT GAC CAG CAC GTT ACC G - 3').

[0145] All PCR products were sequenced by Macrogen Inc. (The Netherlands) in a 96-well plate according to the standard dye terminator sequencing strategy. Sequences were analyzed using BioEdit sequence alignment editor version 7.2.0 (Hall 2007), and multiple sequence alignments were performed using the MAFFT online program (http://mafft.cbrc.iD/aliqnment/software/) (Katoh , Standlev 2013). The sequences were aligned with published NCBI sequences to determine whether any SN Ps were present. The inferred amino acid sequences were used to identify synonymous or non-synonymous mutations.

Population structure and analysis

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37/42 [0146] All samples from the national survey were effectively placed in subpopulations relative to the area in which they were collected. Subpopulations were established by sectioning the country into blocks of 300 x 300 km. The subpopulations were then designated with numbers from 1 to 15 (Supplementary Figure S1). Only subpopulations 2, 7, 8, 11 and 12 were used to determine the population structure due to the lack of sequence data available in the other subpopulations. The population genetic parameters were estimated using POPGENE version 1.31 (Yeh et al. 1997). Qametic imbalance and ancestral recombination [0147] Gametic imbalance studies were performed to determine the intragenic association (link) between SNP alleles within the OCT / Tyr receptor gene. This analysis was performed using the Multilocus 1.2b1 program (Aqapow, Burt 2001). For these analyzes, 100,000 data randomizations were performed to compare the observed data with randomized data that mimic gametic balance. If the observed data set exhibited an increase in gametic imbalance compared to randomized data sets, it was assumed that there is an association between the loci. This was also supported by the P values. Functions performed using Multilocus included analysis of genotypic diversity versus number of loci, linkage imbalance and population differentiation analysis.

[0148] To determine the evolutionary histories within the resistance genes, the ancestral recombination plots were constructed using SNAP Workbench (Price, Carbone 2005). The sequence alignments were converted into haplotypes by excluding indels and infinite location violations. The branching and Beagle algorithm linked in SNAP Workbench was implemented to infer the minimum number of recombination events within the gene that could explain the data (Lynqso, Song, Hein 2005).

Diagnostic assay based on RFLP [0149] The rapid diagnostic test for resistance to amitraz was evaluated in the form of an RFLP analysis. The susceptible R. decoloratus larvae and

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38/42 resistant (confirmed by larval packet tests) were obtained from the University of the Free State, South Africa (Mrs. Ellie van Dalen), for the purpose of a double-blind study. The larvae were labeled from 1 to 14, with their resistance status unknown until after the experiment. The OCT / Tyr receptor gene was amplified from large larval DNA, and amplicons were subsequently treated with 4 U of Ec / 1 restriction enzyme. Depending on the particular pattern that was observed after digestion, the samples were classified as susceptible or resistant to treatment with amitraz. These tests were compared to the resistance states evaluated independently, in order to compare the accuracy of the diagnostic test.

[0150] The aim of this study was to evaluate SNPs at the OCT / Tyr receptor (Chen, He, Davev 2007) in South African field populations of R. microplus. Resistant larvae and susceptible larvae from a controlled and enclosed area in Kruger National Park, South Africa, have been tracked for these two SNPs. A correlation was then made between the presence of these SNPs and LPT results. Consequently, a national survey to detect the presence of these SNPs was then conducted. In order to better understand the complex nature of amitraz resistance in ticks, another analysis was done to test for the presence of gametic imbalance between these SNPs, and to investigate the contribution of recombination towards the emergence of amitraz resistance. Finally, the results of these analyzes led to the development of an RFLP-based diagnostic tool to assess resistance to amitraz in South African tick populations.

[0151] The main objectives of this study were tripled. First, an attempt was made to correlate two known SNPs at the octopaminergic receptor with resistance to amitraz in South African field samples of R. microplus. Second, the gametic imbalance for these SNPs was calculated to determine whether they are randomly associated. Finally, an investigation was launched to assess the evolutionary effects of

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39/42 recombination within the octopaminergic receptor. The results presented here confirmed that the SN Ps of the invention are associated with resistance to amitraz, and that they are in gametic imbalance. Additionally, recombination at the octopaminergic receptor may be correlated with the emergence of resistance to amitraz. These results are useful to farmers in sub-Saharan Africa, and the emergence of resistance to amitraz should be closely monitored in the future. Therefore, the depositor presents a fast and accessible RFLP-based diagnostic technique to assess resistance to amitraz in R. microplus field samples.

4. Conclusion [0152] The invention provides a rapid diagnostic test to assess resistance to amitraz in R. microplus. The test includes the steps of:

extraction of a DNA sample from ectoparasite;

amplification of a DNA sample sequence that corresponds to at least part of an ectoparasite octopamine / tyramine receptor coding sequence;

obtaining an amplification product; and analysis of the amplification product for the presence of one or more markers associated with ectoparasite resistance to acaricide.

Extraction of qenomic DNA from ticks and larvae [0153] A modified salt-based extraction method published by Aljanabi, Martinez (1997) was used to isolate genomic DNA from whole adult ticks (predominantly female ticks). The whole ticks were homogenized in 200 μΙ_ of lysis buffer (0.5 M EDTA, 0.5% (w / v) sodium lauroyl sarcosinate). An additional 400 μΙ_ of DNA extraction solution (0.4 M NaCI, 60 mM Tris-HCI, 12 mM EDTA, 0.25% SDS, pH 8.0) was added to the samples with 2 μΙ_ of proteinase K (15 mg / mL), well mixed and incubated overnight at 55 ° C. The samples were incubated for 20 minutes at 65 ° C to inactivate proteinase K, after which 1 μΙ_ of RNase A (10 mg / mL) was added. The samples were

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40/42 briefly vortexed and further incubated at 37 ° C for 15 minutes. Protein precipitation was performed by adding 360 μΙ_ of 5 M NaCl, vortexing for 10 seconds, and incubating on ice for 5 minutes, followed by centrifugation at 25500 xg for 20 min at room temperature. An equal volume of isopropanol was added to the supernatant, briefly vortexed, followed by a 1 hour incubation at -20 ° C. The samples were then centrifuged for 20 minutes at 10,000 xg and the supernatants discarded. The DNA pellets were washed three times in a row with 500 μΙ_ of 70% ethanol, centrifuged for 5 minutes at 10,000 xg, and the supernatant discarded. The final DNA pellets were air dried and then resuspended in 50 μΙ of 1 x TE buffer (1 mM Tris-HCI, 0.1 mM EDTA, pH 7.0).

[0154] Genomic DNA was extracted from individual larvae using a modification of the protocol by Hernandez et al. (2002). Briefly, the individual larvae were crushed in a 1.5 mL centrifuge tube containing 25 μΙ of TE buffer (10 mM Tris, 1 mM EDTA, pH 7.6). The suspension was then boiled for 5 minutes and centrifuged at 4000 xg for 30 seconds at room temperature. The supernatant was used directly for PCR.

PCR amplification of the octopamine / tyramine receptor [0155] A new direct primer was designed SEQ. ID. AT THE. 1 (OAR-F172 (5'-AGC ATT CTG CGG TTT TCT AC-3 ')) and a SEQ reverse primer. ID. AT THE. 2 (OAR-R587 (5 '- GCA GAT GAC CAG CAC GTT ACC G - 3')) was used for gene amplification.

[0156] New PCR conditions have been developed for amplification, namely:

° C for 4 minutes, followed by 40 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 1 minute, with a final extension of 72 ° C for 8 minutes.

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41/42

The amplification of these genes was done to detect the two published SN Ps:

A22C (nucleotide sequence) / T8P (amino acid sequence)

T65C (nucleotide sequence) / L22S (amino acid sequence) [0157] These two SN Ps were also found with a variety of others. The linkage imbalance studies showed that certain variable positions were closely associated with the two resistant SN Ps, and could therefore potentially be good diagnostic markers of resistance.

Restriction enzyme digestion of amplified gene product with Ecil [0158] (All mutation naming is based on the nucleotide sequences of the coding region only; positions of the non-coding region are excluded).

[0159] One of the mutation sites that are targeted was reported by Chen et al. 2007, but disregarded because it did not result in an amino acid change. The mutation site is C39T. The additional G41A (nucleotide) / G14E (amino acid) mutation site is new and has never been reported before. The chosen enzyme (Ec / 1) will recognize the mutations at both sites. Additionally, the G41A mutation is closely linked to the recombination mutation found at position C159T (coding region), which is also new and is suggested to be the susceptible to resistant switch.

[0160] The amplified fragments of the octopamine receptor were subsequently treated with 4 U of Ec / 1 restriction enzyme in a 50 pL reaction containing 5 pL of CutSmart ™ buffer (50 mM potassium acetate, 20 mM Tris-acetate , 10 mM magnesium acetate and 100 pg / ml BSA, pH 7.9).

[0161] Other enzymes that could be used for diagnosis recognize the 5'-GGATG -3 'sequence, which would take a mutation at the T36C position (nucleotide sequence). This mutation is closely associated with susceptibility rather than resistance, but it could still be used to

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42/42 diagnosis. The list of enzymes includes: BseGI, BstF5l, BstPZ418l, Fokl, Stsl. An enzyme that recognizes the 5'-GGACG -3 'sequence could also be used for the test.

[0162] These changes in the sequence can be detected or diagnosed by sequencing, as well as by RFLP, as mentioned above. It can also be detected with a 'rod' test detection method. The test may include detection of increased enzymes (i.e., glutathione-s-transferases, cytochrome P450s, carboxylesterases). Detection methods for these include qPCR and Taqman assays.

[0163] The test can detect resistance to pyrethroids and formamidines (amitraz). Pyrethroid resistance is well documented. It has already been shown that this test can be applied to Rhipicephalus microplus, Rhipicephalus decoloratus, and other tick species.

[0164] The inventor believes that the invention provides a new method for testing whether a tick has become resistant to amitraz.

Claims (26)

Claims 1. Method to detect a genetic polymorphism associated with susceptibility, or resistance of a tick, in the form of any one or more of Rhipicephalus microplus and Rhipicephalus decoloratus, to amitraz, the method being characterized by understanding the stage of screening a DNA sample of the tick for the presence of one or more markers in the form of single nucleotide polymorphisms (SNPs) in the octopamine / tyramine receptor gene. Method according to claim 1, characterized in that it includes an additional step of determining tick homozygosity and heterozygosity. 3. Method according to claim 1, characterized in that the markers are in the form of any one or more of A22C and T65C, in which the presence of either or both of A22C and T65C is associated with the tick's resistance to amitraz. 4. Method according to claim 1 or 3, characterized in that one or more markers include any one or more among C39T, G41A, G121A, T138C and C159T, in which their presence is associated with the resistance of the tick to amitraz. 5. Method according to claim 1, characterized in that one or more markers include any or both of T36C and G141C, in which the presence of T36C and / or G141C is associated with the susceptibility of the tick to amitraz. 6. Method, according to claim 2, characterized by the determination of homozygosity and heterozygosity of the tick include the classification of ticks having all markers associated with resistance, the markers being single nucleotide polymorphisms (SNP's) in the form of A22C, T65C, C39T, G41A, G121A, T138C and C159T, as homozygous resistant ticks and ticks having only a number of markers associated with resistance, such as heterozygous ticks. Petition 870170044737, of 06/28/2017, p. 48/71 2/6 7. Method, according to claim 1, characterized in that the method includes any suitable technique to determine the presence or absence of one or more markers in the tick's DNA sample, the technique includes any one of: fragment length polymorphism mapping restriction, amplification reactions, nucleic acid hybridization to specific allele probes or oligonucleotide arrays, various chip technologies, polynucleotide sequence techniques and combinations thereof. Method according to claim 1, characterized in that it includes the step of submitting the DNA sample to polynucleotide amplification using a pair of primers comprising SEQ. ID NO. 1 and SEQ. ID. AT THE. 2, and functional fragments, variants, and mutations of each. 9. Method according to claim 1, the method being characterized by comprising the steps of: extracting a DNA sample from the tick; amplification of a sequence of the DNA sample corresponding to at least part of an octopamine / tyramine receptor coding sequence for the tick; obtaining an amplification product; and analysis of the amplification product for the presence of one or more markers associated with susceptibility (T36C and / or G141C) or resistance (A22C, T65C, C39T, G41A, G121A, T138C and / or C159T) of the tick to amitraz. 10. Method according to claim 9, characterized by the step of analyzing the amplification product for the presence of one or more markers associated with susceptibility or resistance of the tick to amitraz by means of any one of: sequencing of the amplification product , quantification of the amplification product, detection of a probe connected to the amplification product and use of a primer extension reaction (PEXT) visualized through a strip immersion test. Petition 870170044737, of 06/28/2017, p. 49/71 3/6 11. Method according to claim 10, characterized by the step of analyzing the amplification product including exposing the amplification product to the restriction enzyme digestion, carried out by using a restriction enzyme having a recognition site corresponding to at least least part of one or more markers. Method according to claim 11, characterized in that the restriction digest is performed by a restriction enzyme having a recognition sequence comprising SEQ. ID. AT THE. 3. Method according to claim 12, characterized in that the restriction enzyme is Ec / 1. 14. Method according to claim 11, characterized in that the restriction is carried out using a restriction enzyme having a recognition sequence comprising SEQ. ID. AT THE. 4. 15. Method according to claim 11, characterized in that the restriction enzyme is selected from the group consisting of any one or more of: BseG \, BstF5 \, BsíPZ418l, Fok \, Sfôl and any enzyme that recognizes a site corresponding to at least part of one or more markers. 16. Method according to claim 11, characterized in that, after digestion, the resulting fragments are separated at least partially from each other using separation techniques based on size. 17. Method according to claim 9, characterized in that DNA is extracted from whole adult ticks using a modified salt-based extraction method for isolating genomic DNA, the method including the steps of: provision of whole tick samples; homogenization of whole ticks in 200 μΙ of lysis buffer (0.5 M EDTA, 0.5% (w / v) sodium lauroyl sarcosinate); addition of an additional 400 μΙ of DNA extraction solution (0.4 M NaCI, Petition 870170044737, of 06/28/2017, p. 50/71 4/6 60 mM Tris-HCI, 12 mM EDTA, 0.25% SDS, pH 8.0) for samples with 2 μΙ of proteinase K (15 mg / ml), well mixed and incubated overnight at 55 ° Ç; incubating the samples for 20 min at 65 ° C to inactivate proteinase K, after which 1 μΙ of RNase A (10 mg / ml) is added; brief submission of the samples to the vortex; incubation of samples at 37 ° C for 15 min; carrying out protein precipitation by adding 360 μΙ of 5 M NaCI, vortexing for 10 sec, and incubating on ice for 5 min, followed by centrifugation at 25 500xg for 20 min at room temperature, to provide a supernatant; addition of an equal volume of isopropanol to the supernatant, brief vortexing, followed by an incubation of 1 hour at -20 ° C; centrifugation of the samples for 20 min at 10 OOOxg and disposal of supernatants; washing DNA pellets three times in a row with 500 μΙ of 70% ethanol, centrifuged for 5 min at 10 OOOxg, and discarding the supernatant; air drying the final DNA pellets; and resuspension of air-dried DNA pellets in 50 μΙ of 1 x TE buffer (1 mM Tris-HCI, 0.1 mM EDTA, pH 7.0). 18. Method according to claim 9, characterized by the Genomic DNA being extracted from the individual larvae, the extraction method including: crush the individual larvae in a 1.5 ml microcentrifuge tube containing 25 μΙ of TE buffer (10 mM Tris, 1 mM EDTA, pH 7.6) to form a suspension; boil the suspension for 5 min; centrifuge at 4000xg for 30 sec at room temperature to provide a supernatant; and use the supernatant for PCR. Petition 870170044737, of 06/28/2017, p. 51/71 5/6 19. Method according to claim 9, characterized by the step of amplifying a sequence of the DNA sample to be carried out by submitting the DNA sample to the following conditions of heating, annealing and cooling, respectively: heat to 94 ° C for 4 min; followed by 40 cycles of 94 ° C for 30 sec, 55 ° C for 30 sec and 72 ° C for 1 min; and with a final extension of 72 ° C for 8 min. 20. Isolated nucleic acid molecule in the form of c-DNA, characterized by being selected from the group consisting of: (i) a nucleic acid molecule comprising the SEQ sequence. ID. AT THE. 1; (ii) a nucleic acid fragment having a sequence derived from an octopamine / tyramine receptor gene and containing any one or more of the markers associated with tick resistance to an amitraz, the markers selected from: A22C, T65C, C39T, G41A , G121A, T138C and C159T; (iii) a nucleic acid fragment having a sequence derived from an octopamine / tyramine receptor gene and containing any one or more of the markers associated with a tick's susceptibility to an amitraz, the markers selected from: T36C and G141C; and (iv) complementary sequences. 21. Amplified polynucleotide, characterized by having a polymorphism in the form of any one or more of: A22C, T65C, C39T, G41A, G121A, T138C, C159T, T36C and G141C. 22. Use of an isolated nucleic acid molecule, as defined in claim 20 or an amplified polynucleotide, as defined in claim 21, characterized in that it is in a method for detecting a genetic polymorphism associated with susceptibility or resistance of a tick to amitraz. Petition 870170044737, of 06/28/2017, p. 52/71 6/6 23. Use of any one or more of the genetic markers C39T, G41A, G121A, T138C and C159T, characterized by being indicators of resistance of a tick to an amitraz. 24. Use of either or both of the genetic markers T36C and G141C, characterized by being as indicators of susceptibility of a tick to an amitraz. 25. Use of any one or more of the genetic markers in the form of G14E, T8P and L22S peptide-based markers, characterized as being indicators of tick resistance to an amitraz. 26. Kit for determining susceptibility, or otherwise of a tick to amitraz, the kit characterized by including: primers in the form of SEQ. ID. NO.1 and SEQ. ID. AT THE. 2; and a suitable reagent buffer to allow amplification of a DNA sequence to be amplified using the primers.