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.


Introduction
Rhipicephalus (B.) decoloratus is a one-host tick that is widely distributed in Africa [1][2][3]. This tick is an important vector for Babesia spp. and Anaplasma spp., which causes bovine babesioisis and anaplasmosis, respectively [4,5]. The burden and importance of the R. (B.) decoloratus tick have been scantly reported in Uganda [6][7][8]. Chemicals (acaricides) are the common means for tick control and prevention of the diseases vectored by ticks. The emergence of resistance against synthetic pyrethroids (SP) and its co-formulations with organophosphates (OP) makes amitraz a cornerstone for tick control in Uganda. Amitraz is one of the most widely used acaricides for tick control in Uganda [9,10] and in the rest of Africa [11][12][13]. Amitraz belongs to a group of pesticides known as formamidines [14,15]. Formamidines achieve their pharmacological activity by modulating tick octopamine receptors [16,17]. The octopamine receptor is a member of the G-protein coupled receptor (GPCR) that is analogous to the adrenergic receptors in vertebrates [18]. The arthropod octopamine receptors are classified into three, namely alpha (α) and beta (β) adrenergic-like and octopamine/tyramine receptors [19][20][21]. Amitraz causes impairment of tick nervous functions of octopamine and it inhibits oviposition [22]. This unique mode of action of amitraz makes it key in the acaricide rotation against ticks that are resistant to SP and OP acaricides. While there are few reports on amitraz resistance in R. (B.) decoloratus, on the contrary, amitraz resistance is widely reported in R. (B.) microplus from Australia, South America, India and South Africa [12,[23][24][25]. The genetic basis of amitraz resistance in R. (B.) microplus was first attributed to mutations in the octopamine receptor [26]. This was confirmed by researchers in South Africa who found that mutations in the octopamine/tyramine (OCT/Tyr) gene, confered resistance against amitraz in South African R. (B.) microplus tick strains [12]. The same mutation in R. (B.) microplus OCT/Tyr that confered amitraz resistance was also associated with amitraz resistance in R. (B.) decoloratus ticks from South Africa [12].
In Uganda, we previously reported the emergence of amitraz-resistant R. (B.) decoloratus ticks in western cattle corridor [27]. However, there is lack of data on a molecular basis of amitraz resistance in Ugandan strains of R. (B.) decoloratus ticks. Genetic techniques have been widely used to investigate the mechanisms of resistance in arthropod vectors [28][29][30]. In the present study, we characterized the R. (B.) decoloratus OCT/Tyr receptor gene from both amitraz-susceptible and -resistant larval tick populations and identified novel mutations that are associated with phenotypic resistance against amitraz in tick populations from Uganda.

Tick Population
The ticks used in this study were partly retrieved from archived samples previously collected from cattle farms in Uganda [27,31]. A total of 17 R. (B.) decoloratus larval tick populations were used. The ticks were categorized into two, based on the region and intensity of acaricide use. The first group were those from farms in central (2) and western Uganda (13) referred to as ticks from high acaricide pressure area. The second category was an amitraz-susceptible population collected from eastern (01) and northern (01) Uganda in an area of low acaricide pressure. The districts from which tick samples were collected is shown in Figure 1.
The farms in the high acaricide pressure region mainly kept crosses of exotic dairy cattle that are susceptible to tick-borne diseases, while those from the low acaricide pressure region kept indigenous cattle breed (Zebu cattle). The engorged female ticks collected from the above farms were reared at the Research Center for Tropical Diseases and Vector Control (RTC) Laboratory, College of Veterinary Medicine, Animal Resources and Biosecurity (COVAB), Makerere University. The susceptibility of the larvae against amitraz was determined by the Larval Packet Test (LPT) as previously reported [27]. The characteristics of the farms and tick populations investigated is shown in Table 1.

Extraction of Genomic DNA
For each tick population, genomic DNA was extracted from approximately 30 pooled larvae from using a Nucleospin Tissue DNA extraction kit (Marcherey-Nagel, Duren, Germany). The larvae that were preserved in 75% ethanol were washed with 1 × PBS and crushed in a Bio-masher II tube (Nippi, Japan) with Bio-masher motor (Nippi, Japan). The DNA was extracted using the above kit following the manufacturer's instructions. The concentration of the extracted DNA was determined with a Nano Drop 2000 (Thermo Fisher Scientific Inc, Waltham, MA, USA) and a working stock of 40 ng/µL was prepared and stored at −30 • C until use. All the DNA samples used had a purity ratio of above 1.8. The control DNA from R. (B.) microplus was extracted from adult ticks collected from north-eastern Thailand archived at the National Research Center for Protozoan Diseases (NRCPD), Obihiro University of Agriculture and Veterinary Medicine-Japan.  LPT, larval packet test; Both (Local and improved cattle). Þ No amitraz used in the last 2 years; Þ Control populations from low acaricide pressure area of Uganda, (-) not disclosed; Tick populations used in this study were archived samples and amitraz susceptibility was determined in our previous study [27,31]. AM, amidine (amitraz); OP, organophosphate (chlorfenvinphos); SP, synthetic pyrethroid (cypermethrin or deltamethrin); COF, co-formulation containing organophosphate (chlorfenvinphos or chlorpyriphos) and synthetic pyrethroid (cypermethrin).

Amplification of Octopamine/Tyramine Receptor Gene
The octopamine/tyramine receptor gene was amplified using a Blend Taq ® plus polymerase kit (Toyobo, Japan). The primers designed by previous researchers were used to amplify the region of the OCT/Tyr gene that is susceptible to mutation [26]. These primers were OAR-F171: 5 -GGTTCACCCAACCTCATCTCTGAA-3 (forward) and OAR-R587: 5 -GCAGATGACCAGCACGTTACCG-3 (reverse). Amplification was carried out in a 40 µL reaction volume containing 0.2 mM dNTP, 0.24 mM of each primer and 2 ng of genomic DNA template. The thermal cycling conditions included 1 min of initial denaturation at 94 • C and 40 cycles of 94 • C for 5 s and 55 • C for 1 min and further extension of 68 • C for 5 min. The resultant PCR products were electrophoresed in 1.5% agarose gel, stained with ethidium bromide and visualized under UV lamp.

Cloning and Sequencing of Octopamine/Tyramine Receptor Gene
The OCT/Tyr PCR amplicons were purified with Wizard ® SV Gel and a PCR Clean-up System (Promega, Madison, WI, USA) according to the manufacturer's instruction. The gene was ligated in pGEM-Teasy (Promega, Madison, WI, USA) at 4 • C overnight and transformed into ECOS TM competent Escherichia coli DH5α (Nipon gene, Tokyo, Japan). For each sample, successful clones were confirmed by colony PCR and at least 4 colonies were multiplied in Luria-Bertaini broth (with 100 µg/mL ampicillin) and purified using a NucleoSpin ® Plasmid Easy Pure kit (Marcherey-Nagel, Duren, Germany) following the manufacturer's instruction. The OCT/Tyr gene insert was sequenced with T7 promoter (forward) primer using a BigDye v3.1 Terminator Cycle Sequencing Kit and the 3730 × l DNA Analyzer (Applied Biosystem, Waltham, MA, USA).

Sequence Analysis
The nucleotide sequence was edited with DNA star (Version 7.1.0) and the resultant sequence was analyzed with BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi, (last accessed on 30 November 2022) to determine percentage identity. Mutations in the coding region of the OCT/Tyr gene was analyzed by multiple sequence alignment of the corresponding nucleotide and amino acid sequences using GeneDoc ver 2.7.000. Nucleotide diversity in both acaricide-susceptible and -resistant ticks was compared and their evolutionary history was determined using MEGA5.0 software [32]. The resultant R. (B.) decoloratus Oct/tyramine nucleotide sequences generated in this study were deposited in GenBank under the Accession # ON792209-ON792255.    (Figure 3).

Analysis of SNPs in the Coding Region of OCT/Tyr Receptor Gene
The R. (B.) decoloratus OCT/Tyr partial ORF revealed variable levels of nucleotide similarity ranging from 93.1-100.0% among the different clones and tick populations. Overall, 32 single nucleotide polymorphism (SNPs) were recorded amongst R. (B.) decoloratus OCT/Tyr gene sequences in this study. However, 24 (75%) of the above mutations were only found in ticks from high acaricide pressure areas (HAPA) of western and central Uganda. In contrast, only 8 (25%) SNPs were found in the OCT/Try receptor gene from the R. (B.) decoloratus ticks collected from low acaricide pressure areas (LAPA) in north and eastern Uganda. Only two SNPs were shared between OCT/Tyr genes from ticks collected from Serere (LAPA) and HAPA. Eleven of the above SNPs resulted in a change of amino acid (non-synonymous) in the OCT/Tyr receptor gene (Table 2).
Novel non-synonymous SNPs were observed at four different loci in the ORF for each of the four R. (B.) decoloratus resistant tick populations; A1G (KMGR), C46T (2MTM), A122G (1BUS) and T215C (3MTM) (Figure 3). Based on previous nomenclature, A1G, C46T, A122G and T215C corresponds to A135G, C181T, A257G and T350C, respectively [12,26]. Two additional identical non-synonymous SNPs, C215T and C230T were also found in the R. (B.) decoloratus OCT/Tyr gene from the HAPA with no particular pattern. However, all the non-synonymous SNPs in OCT/Tyr from the amitraz-susceptible R. (B.) decoloratus ticks were not related to those from the high acaricide pressure area as shown in Table 2 above.

OCT/Tyr Gene Amino Acid Mutations in Amitraz-Resistant Ticks
Overall, the R.  Four of the amino acids (M1V, L16F, D41G, V72A) out of the eight mutations corresponded to the novel non-synonymous SNPs found amongst amitraz-resistant tick populations from western Uganda. The amino acid mutation at position 16 (leucine to phenylalanine) was associated with 58.5% survival amongst pooled larvae from farm 2MTM as previously reported by our research team [27]. Low survival rate of 31.9% was associated with mutation at position 41, which led to a change from aspartate to glycine (D41G) in ticks from farm 1BUS. However, amino acid substitution at position 72 (valine to alanine) was associated with 55% survival of larvae against amitraz in tick population from farm 3MTM. Two identical amino acid substitutions at position 27 and 32 (valine to alanine) that corresponds to the C215T and C230T SNPs were recorded in the R. (B.) decoloratus OCT/Tyr gene from the high acaricide pressure area. Interestingly none of the amino acid mutations in the R. (B.) decoloratus OCT/Tyr gene from the susceptible tick populations were related to those from the high acaricide pressure area (Table 2).

Discussion
We previously reported the emergence of the amitraz-resistant tick population in the greater Bushenyi area in Uganda through phenotypic assays. Amitraz is considered as a cornerstone in the control of resistant ticks because it is the choice of acaricide for control of orgnophosphate-and pyrethroid-resistant ticks given its unique mode of action. The OCT/Tyr homologue reported in this study is one of the target sites for amitraz according to previous studies [12,19]. The current study found greater homology between the R. The high number of non-synonymous SNPs in the R. (B.) decoloratus OCT/Tyr gene from the western region is suggestive of selection pressure caused by frequent exposure of ticks to amitraz. Selection pressure increases the rate of resistance alleles in ticks [23]. Up to eight amino acid substitutions were observed in the OCT/Tyr gene from amitrazresistant ticks from western Uganda. However, four of the above substitutions, M1V, L16F, D41G and V72A, were strictly found in the R. (B.) decoloratus tick populations that were resistant to amitraz. Overall, all the amino acid mutations observed in this study were unique from those previously reported in amitraz-resistant R. (B.) microplus. Chen and his colleagues first reported the T8P and L22S as resistance SNPs, which was later confirmed by other researchers in South Africa, India and Brazil [12,26,33,34]. However, this contrasts with our findings in which each of the resistant tick populations had more than one SNP loci that were not related to previous amino acid substitutions reported in South Africa [12]. What remains unclear is whether the various non-synonymous SNPs identified in amitraz-resistant ticks play a key role in amitraz resistance in R. (B.) decoloratus ticks from Uganda.
The L16F mutation was associated with 58.5% amitraz survival by LPT and history of consistent use of amitraz once a week for 2 years. However, D41G and V72A mutation was associated with 31.9% amitraz survival and intermittent use of amitraz. Interestingly, the V72A mutation was associated with 55% amitraz survival rate, yet the history of acaricide use showed that the farmer did not use amitraz for the last 2 years (Table 1). This could be attributed to new animals brought on the farm with ticks that were resistant to amitraz, or other factors that could introduce resistant ticks into the farm. We previously reported that cattle trade and the lapse in inspection and quarantine of purchased animals is amongst the risk factors for spreading acaricide-resistant ticks in Uganda [10,27].
The current study also found two identical amino acid substitutions (V to A) at two loci, 27 and 32, in the OCT/Tyr genes of R. (B.) decoloratus ticks from western Uganda. There were no particular patterns in the occurrence of the above mutations between resistant and susceptible genotypes from the high acaricide pressure area. Surprisingly, one of the tick populations (KKS) that showed 100% survival at a discriminating dose of amitraz did not have unique amino acid substitutions in the OCT/Tyr receptor. This further provides hints that amitraz resistance in Boophilus ticks may be mediated via multiple pathways, with OCT/Tyr target site mutation being possibly one of the several mechanisms. Several researchers have highlighted the potential role of other G-protein coupled receptors such as beta-and alpha-adrenergic-like-octopamine receptors and various metabolic enzymes in amitraz-resistant Rhipicephalus ticks [12,33,35]. Indeed researchers from South Africa previously identified several transcripts of enzymes from amitraz-resistant ticks that were upregulated following exposure to amitraz [36]. This implores the need for further investigations to understand the mechanism of amitraz resistance using both functional genomics and metabolomics.
In the current study, phylogenetic analysis revealed that the Ugandan strain of R. (B.) decoloratus OCT/Tyr genes clustered into two distinct groups that separated the genes from high acaricide pressure areas (central and west) from the susceptible populations (east and north). Of concern was the interspersal of resistant genotypes within the susceptible ones in the high acaricide pressure areas. This could imply that the non-synonymous SNPs observed in the OCT/Tyr receptor coding region is an early indicator for potential amitraz resistance development. It should be noted that there has been prolonged irrational use of various acaricides, including amitraz in Uganda. Therefore, to gain more understanding about the role OCT/Tyr receptor in amitraz resistance in R. (B.) decoloratus ticks, we recommend further research using a large number of amitraz-resistant tick populations with various levels of resistance. However, at the farm level, we recommend farmer training on proper acaricide application and rotation across the country. This will help to preserve the efficacy of amitraz, given that resistance to OP and its co-formulation with synthetic pyrethroids is wide spread in Uganda [27].
In conclusion, this study characterized the R. (B.) decoloratus OCT/Tyr receptor gene from both amitraz-susceptible and -resistant Ugandan tick strains. For the first time, we report four unique amino acid substitutions in the R. (B.) decoloratus OCT/Tyr receptor gene that are associated with phenotypic resistance against amitraz, although we postulate that other mechanisms of resistance may play a significant role in amitraz resistance. The finding from this study is expected to stimulate further research on the molecular and biochemical basis of amitraz resistance in R. (B.) decoloratus ticks in Africa and beyond.  Data Availability Statement: The nucleotide sequences generated in this study have been deposited in GenBank for public access.