(Boophilus) Microplus 68 4 69 A, B, 2 C B C 5 Q5 Aaron D

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55 Contents lists available at ScienceDirect 56 57 Insect Biochemistry and Molecular Biology 58 59 60 journal homepage: www.elsevier.com/locate/ibmb 61 62 63 64 65 1 Pharmacological characterization of a tyramine receptor from the 66 2 67 3 Q6 southern cattle tick, Rhipicephalus (Boophilus) microplus 68 4 69 a, b, 2 c b c 5 Q5 Aaron D. Gross , Kevin B. Temeyer , Tim A. Day , Adalberto A. Perez de Leon , 70 * 6 Michael J. Kimber b, 1, Joel R. Coats a, , 1 71 7 72 8 a Pesticide Toxicology Laboratory, Department of Entomology, Iowa State University of Science and Technology, Ames IA 50011, USA b 73 9 Department of Biomedical Science, Iowa State University of Science and Technology, Ames IA 50011, USA c Knipling-Bushland United States Livestock Insects Research Laboratory and Veterinary Pest Genomics Center, United States Department of Agriculture- 74 10 Agricultural Research Service, Kerrville, TX 78028, USA 75 11 76 12 77 13 article info abstract 78 14 79 15 Article history: The southern cattle tick (Rhipicephalus (Boophilus) microplus) is a hematophagous external parasite that 80 16 Received 16 February 2015 vectors the causative agents of bovine babesiosis or cattle tick fever, Babesia bovis and B. bigemina, and 81 17 Received in revised form anaplasmosis, Anaplasma marginale. The southern cattle tick is a threat to the livestock industry in many 82 21 April 2015 18 locations throughout the world. Control methods include the use of chemical acaricides including Accepted 27 April 2015 83 amitraz, a formamidine insecticide, which is proposed to activate octopamine receptors. Previous studies 19 Available online xxx 84 fi 20 have identi ed a putative octopamine receptor from the southern cattle tick in Australia and the 85 Americas. Furthermore, this putative octopamine receptor could play a role in acaricide resistance to 21 Keywords: 86 amitraz. Recently, sequence data indicated that this putative octopamine receptor is probably a type-1 22 Tyramine receptor 87 23 Southern cattle tick tyramine receptor (TAR1). In this study, the putative TAR1 was heterologously expressed in Chinese hamster ovary (CHO-K1) cells, and the expressed receptor resulted in a 39-fold higher potency for 88 24 G-protein-coupled receptor GPCR tyramine compared to octopamine. Furthermore, the expressed receptor was strongly antagonized by 89 25 Tick yohimbine and cyproheptadine, and mildly antagonized by mianserin and phentolamine. Tolazoline and 90 26 Rhipicephalus microplus naphazoline had agonistic or modulatory activity against the expressed receptor, as did the amitraz 91 27 metabolite, BTS-27271; however, this was only observed in the presence of tyramine. The southern cattle 92 28 tick's tyramine receptor may serve as a target for the development of anti-parasitic compounds, in 93 29 addition to being a likely target of formamidine insecticides. 94 © 30 2015 Published by Elsevier Ltd. 95 31 96 32 97 33 98 34 99 35 1. Introduction of vectoring the apicomplexan protozoa Babesia bovis and B. bige- 100 36 mina, which cause bovine babesiosis, and the rickettsia Anaplasma 101 37 The southern cattle tick (Rhipicephalus (Boophilus) microplus)is marginale that causes anaplasmosis (Perez de Leon et al., 2012; 102 38 an ectoparasite that can have an economically devastating effect on Perez de Leon et al., 2014). 103 39 the cattle industry worldwide (Grisi et al., 2014). R. microplus, in the The United States initiated the Cattle Fever Tick Eradication 104 40 United States, was estimated to have direct costs of more than $130 Program (CFTEP) in 1906, which eliminated the southern cattle tick 105 41 million to the cattle industry in 1906, which has been estimated to (R. microplus) and the cattle fever tick (R. annulatus) from thirteen 106 42 be over $3 billion today (USDA-APHIS August 2010). The significant southern states by 1941 (Perez de Leon et al., 2012). The mainstay of 107 43 economic impact of this tick is realized with the diseases that it is the CFTEP is the use of chemical acaricides; specifically, the organ- 108 44 capable of vectoring. Specifically, the southern cattle tick is capable ophosphate coumaphos. However, internationally several chemical 109 45 acaricides, with different mechanisms of action, have been used to 110 46 control the southern cattle tick, and has predictably resulted in 111 47 112 * Corresponding author. Iowa State University, Department of Entomology, 116 acaricide resistance. Acaricide resistance, particularly with tick 48 Insectary Building, Ames, IA 50011, USA. Tel.: þ1 515 294 4776. strains found in the Americas, has been reported for several chemical 113 49 E-mail address: [email protected] (J.R. Coats). classes including organophosphates (Miller et al., 2005; Rodriguez- 114 50 1 Co-senior authors. 115 2 Vivas et al., 2007; Temeyer et al., 2007; Miller et al., 2008; Guerrero 51 Current address: Emerging Pathogens Institute, Department of Entomology and 116 Nematology, University of Florida, Gainesville, FL, 32610, USA. et al., 2012), synthetic pyrethroids (Li et al., 2007; Miller et al., 2007a; 52 117 53 http://dx.doi.org/10.1016/j.ibmb.2015.04.008 118 54 0965-1748/© 2015 Published by Elsevier Ltd. 119

Please cite this article in press as: Gross, A.D., et al., Pharmacological characterization of a tyramine receptor from the southern cattle tick, Rhipicephalus (Boophilus) microplus, Insect Biochemistry and Molecular Biology (2015), http://dx.doi.org/10.1016/j.ibmb.2015.04.008 IB2701_proof ■ 9 May 2015 ■ 2/7

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1 Cossio-Bayugar et al., 2008; Guerrero and Nene, 2008; Rosario-Cruz octopamine hydrochloride (99%), (±)-synephrine, dopamine hy- 66 2 et al., 2009) and formamidines (Li et al., 2007; Miller et al., 2007b; drochloride (98.5%), DL-norepinephrine hydrochloride (97%), 67 3 Corley et al., 2013). Recently, a population of R. microplus of serotonin hydrochloride (5-hydroxytryptamine, 5-HT), 68 4 concern has been discovered in Mexico. This population is resistant (±)-epinephrine hydrochloride, histamine dihydrochloride (99%), 69 5 to synthetic pyrethroids, organophosphates, amitraz (formamidine) phentolamine hydrochloride (98%), yohimbine hydrochloride 70 6 and ivermectin (macrocyclic lactone family) (Fernandez-Salas et al., (98%), clonidine hydrochloride (solid), mianserin hydrochloride, 71 7 2012). The permanent quarantine zone in south Texas along the (±)-propranolol hydrochloride (99%), chlorpromazine hydro- 72 8 TexaseMexico border is part of the continued effort by the CFTEP to chloride (98%), amitraz metabolite BTS-27271 (N-(2,4- 73 9 keep the U.S. free of cattle fever ticks and bovine babesiosis. As Dimethylphenyl)-N0-methylformamidine, N-Methyl-N0-2,4- 74 ® 10 R. microplus is endemic to Mexico, there is the possibility of regular xylylformamidine) Pestanal (Fluka), naphazoline hydrochloride, 75 11 reintroduction of the southern cattle tick into the U.S. Reintro- 2-benzylimidazoline (tolazoline). 76 12 duction is possible with the movement of cattle into the U.S. from Test compounds were prepared fresh for each testing procedure 77 13 Mexico and the ability of the tick to be maintained onwild ungulates. with the exception of the amitraz metabolite BTS-27271, which was 78 14 For continued success of the CFTEP, it is imperative that integrative used from frozen DMSO stocks. Tyramine and octopamine were 79 15 pest control measures are taken, that new biochemical targets for prepared in Hank's Balance Salt Solution (HBSS, Life Technologies, 80 16 tick control are identified and a greater understanding of the Carlsbad, CA). The remaining compounds were dissolved in DMSO 81 17 mechanism(s) of acaricide resistance (Temeyer et al., 2012; Perez de then serially diluted with HBSS. The cells were exposed to a final 82 18 Leon et al., 2012; Guerrero et al., 2014). concentration of 0.1% DMSO. 83 19 Octopamine and tyramine are biogenic amines found in a many 84 20 of invertebrates, including insects and ticks, and have been shown 2.2. Putative tyramine receptor 85 21 to be involved in different physiological functions in insects. These 86 22 physiological functions are realized when octopamine or tyramine The wild-type putative tyramine receptor (EF490687.1) was 87 23 activates their cognate receptor, which are G-protein-couple re- isolated from the Gonzalez strain of tick (amitraz-susceptible). The 88 24 ceptors (GPCRs) (Guerrero and Dowd, 2010). Octopamine and Gonzalez strain of R. microplus was originally identified in Zapata 89 25 tyramine are characterized as neurotransmitters, neurohormones County, Texas in 1984, and has been maintained in culture by the 90 26 and neuromodulators (Roeder et al., 2003; Roeder, 2005; Farooqui, USDA-ARS Knipling-Bushland U.S. Livestock Insects Research Lab- 91 27 2012; Ohta and Ozoe, 2014). While the diverse physiological oratory (KBUSLIRL), Kerrville, TX at the USDA-ARS Cattle Fever Tick 92 28 functions of octopamine have been realized for some time, we are Research Laboratory (CFTRL), Edinburg, TX (Chen et al., 2007). The 93 29 only starting to understand the physiological relevance of tyra- receptor cDNA was synthesized by GeneScript USA Inc. (Piscataway, 94 30 mine; this is because, until recently, tyramine was thought to only NJ) and cloned into a pCDNA3.1() expression vector (Life 95 31 be a synthetic precursor to octopamine (Guerrero and Dowd, 2010; Technologies). 96 32 Roeder et al., 2003; Roeder, 2005; Verlinden et al., 2010; Farooqui, 97 33 2012). Octopamine receptors are believed to be the target of for- 2.3. CHO cell culture, transfection and calcium mobilization assay 98 34 mamidine insecticides/acaricides, which include chlordimeform 99 35 and amitraz (Blenau et al., 2012; Hashemzadeh et al., 1985). Hy- Cell culture materials were obtained from Life Technologies, 100 36 peractivity and tick detachment are symptoms of exposure to sub- unless otherwise stated. Chinese hamster ovary-K1 (CHO or CHO- 101 37 lethal concentrations of formamidine acaricides against the K1) cells were obtained from American Type Culture Collection 102 38 southern cattle tick (Stone et al., 1974). (ATCC; Manassas, VA). CHO cells were maintained in a NuAire hu- 103 39 A cDNA sequence (AJ010743.1), originally thought to code for an midified water jacket incubator (Plymouth, MN); the incubator was 104 40 octopamine receptor, was isolated from two strains of R. microplus maintained at normal cell growth conditions of 37 C, 90% humidity 105 41 ticks that were susceptible and resistant to amitraz in Australia. and 5% CO2. The normal cell growth medium consisted of 1X Ham's 106 42 However, the identified open reading frame (ORF) did not result in F12K (Kaighn's modification) medium supplement with 10 mM 107 43 any point mutations (Baxter and Barker, 1999). The putative HEPES (N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid) 108 44 octopamine receptor was later identified in tick populations from plus 10% fetal bovine serum (FBS) (Atlas Biologicals, Fort Collins, 109 45 the Americas (EF490687.1 and EF490688.1) (Chen et al., 2007). CO). CHO cells were subcultured when cell confluency reached 110 46 Additionally, two point mutations, T8P and L22S, were identified in 80%e90%. To subculture, cells were washed with Dulbecco's 111 47 this putative receptor's open reading frame obtained from amitraz- Phosphate Buffered Saline (DPBS); then cells were detached from 112 48 resistant ticks (EF490688.1). These point mutations were hypoth- culturing flasks with 0.25% trypsin e 0.53 mM EDTA (trypsin-EDTA) 113 49 esized to have a possible role in the resistance to the acaricide solution. Cells were then centrifuged at 900 g for 5 min at room 114 50 amitraz (Chen et al., 2007). However, the functional identification temperature; cell pellet was resuspended in fresh normal cell 115 51 of these receptors ligands has yet to be performed. In fact, it has growth media. 116 52 been hypothesized, based on sequence similarity, that the origi- Transient transfection of CHO cells with the putative tyramine 117 53 nally identified receptor is a tyramine receptor, instead of an receptor was performed in 6-well cell culture plates (Corning). Cells 118 ® 54 octopamine receptor (Verlinden et al., 2010; Farooqui, 2012). The were transfected using Lipofectamine 2000 in OPTI-MEM sup- 119 55 objectives of this study were to (1) functionally characterize the plement with F12K þ 10% FBS, according to the manufacturer's 120 56 putative tyramine receptor from the southern cattle tick, and (2) protocols (Invitrogen). Cells were transfected with 2.5 mg of vector 121 57 determine the receptor pharmacology using functional assays. DNA, which allowed the examination of the putative tyramine re- 122 58 ceptor's ability to link to the endogenous calcium pathway (Gaq). 123 59 2. Materials and methods CHO cells were also transfected with individual G-protein chimeras 124 60 (Gaqo5,Gaqi5,Gaqs5,orGaqz5) to force receptor-mediated signaling 125 61 2.1. Chemicals down the calcium-signaling pathway as previously described 126 62 (Conklin et al., 1996; Omar et al., 2007). CHO cells were incubated in 127 63 Pharmacological analysis was performed with compounds the transfection medium for 12 h at normal cell growth conditions. 128 64 purchased from SigmaeAldrich (St. Louis, MO) unless otherwise After 12 h, cells were lifted from 6-well plate using trypsin-EDTA. 129 65 noted. Test compounds include tyramine hydrochloride (98%), DL- Cells were washed with normal growth medium and moved to a 130

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1 15-mL conical tube, and were centrifuged at 900 g at room signal using the Gaq PLC pathway; therefore, a set of Gaq-protein 66 2 temperature for 5 min. The cell pellet was resuspended in 3e3.5 mL chimeras, which funnel receptor activation down the Gaq PLC 67 3 of normal cell growth medium. CHO cells were counted using a pathway resulting in an increase of intracellular calcium, were used 68 ® 4 Countess Automatic Cell Counter (Life Technologies), according to (Fig. 1). These G-protein chimeras were developed to have the 69 5 the manufacturer's protocols. CHO cells were placed into black-wall receptor-interacting domain (5 amino acids) from different 70 6 clear-bottom 96-well plates (Corning) at a cell density of 27,500 G-proteins (Gas,Gaz,Gai and Gao), built on the Gaq backbone to 71 7 cells per well, with a total volume of 100 mL. Cells were incubated at make a hybrid calcium-sensitive signaling molecule designated as 72 8 normal cell culture conditions for 24 h prior to performing the Gaqs(5),Gaqz(5),Gaqi(5) and Gaqo(5). Therefore, heterologous 73 9 calcium mobilization assay. The calcium mobilization assay was expression of a receptor with the Gaq chimera allows for quantifi- 74 10 performed with a Fluo-4 NW Calcium Assay Kit (Life Technologies), cation of receptor activation using a fluorescence output (Fig. 1). 75 11 according to the manufacturer's protocol. Briefly, the cells were The R. microplus putative tyramine receptor was transiently 76 12 incubated in HBSS, 20 mM HEPES, a calcium-sensitive fluorescent expressed in CHO cells alone to see if the receptor activated the 77 13 dye (Fluo-4) and 2.5 mM probenecid; cells were incubated in this endogenous Gaq PLC pathway (Fig. 1). Expression of the receptor 78 14 medium for 90 min at 37 C prior to the start of the assay. Fluo- alone resulted in a weak fluorescent response to tyramine and 79 15 rescence was monitored using a FlexStation 1 (Molecular Devices, octopamine at 1 mM(Fig. 1). Transient expression was performed 80 16 Sunnyvale, CA). Fluorescence changes were monitored with exci- with the putative tyramine receptor in combination with individual 81 17 tation at 494 nM and emission at 516 nM. Basal fluorescence was Gaq chimeras (Gaqs(5),Gaqz(5),Gaqi(5) and Gaqo(5)). As shown in 82 18 monitored for 20 s prior to the addition of screening compounds. Fig. 1, co-expression of the putative tyramine receptor resulted in 83 19 The fluorescence readings were measured every 1.5 s for 120 s. the most significant response when co-expressed with the Gaqi(5) 84 20 Stable transfection was performed using the transient trans- G-protein chimera (Fig. 1). Additionally, 1 mM tyramine had a 85 21 fection procedure. However, instead of proceeding to the functional stronger and more significant fluorescence response when 86 22 assay, cells were maintained in medium containing 100 mg/mL of compared to 1 mM octopamine, which indicated that tyramine 87 ® 23 Geneticin (G418 sulfate; Corning). Single-cell colonies were preferentially activated the heterologously expressed receptor with 88 24 selected in 96-well plates over four weeks. Successfully transfected the Gaqi(5) G-protein chimera. Tyramine and octopamine displayed 89 25 cells, with both the putative tyramine receptor and G-protein concentration-dependent responses in CHO cells transiently 90 26 chimera, were determined with a calcium mobilization assay and expressing the receptor along with the Gaqi(5) chimeric protein 91 27 by RT-PCR. (Fig. 2). Tyramine had an EC50 of 9 nM, whereas octopamine had an 92 28 Compounds were screened using an agonist and/or an antago- EC50 of 351 nM; therefore, tyramine is 39-fold more effective at 93 29 nist screening method. The agonist screening method was utilized activating the heterologously expressed receptor with the Gaqi(5) 94 30 with the addition of the potential agonist added by the FlexStation chimera, compared to octopamine (Fig. 2 and Table 1). Additional 95 31 after a 20-second background reading. Antagonist or modulator biogenic amines were screened at 1 mM to ensure that tyramine is 96 32 screening was performed by preincubating the putative antagonist the preferred ligand. Tyramine activated the receptor most signif- 97 33 or modulator with the cells in the black-walled clear-bottom 96- icantly, followed by octopamine. Acetylcholine, along with the N- 98 34 well plate for 30 min prior to the addition of tyramine (addition methylated analogue of octopamine, synephrine, were capable of 99 35 performed by the FlexStation). mildly activating the expressed receptor and Gaqi(5) chimera, but 100 36 not as significantly as tyramine or octopamine (Fig. 3). Dopamine, 101 37 2.4. Data analysis 5-HT (serotonin), norepinephrine, epinephrine and histamine had 102 38 little effect against the expressed receptor and chimera at 1 mM 103 39 A minimum of six wells per plate along with a minimum of four (Fig. 3). These results indicated that the originally classified 104 40 plates was analyzed for each treatment. Additionally, each plate 105 41 contained several controls: a blank (calcium fluorescent indicator, 106 42 probenecid (2.5 mM) and HBSS with 10 mM HEPES buffer), a 107 43 vehicle control, along with an autofluorescent control (fluorescent 108 44 indicator (Fluo-4), HBSS with 10 mM HEPES and the ligand). 109 45 Additionally, non-receptor mediated effects were monitored for 110 46 each treatment and consisted of untransfected cells not expressing 111 47 the receptor (regular CHO cells). Concentration-response curves 112 48 were analyzed in GraphPad Prism (La Jolla, CA). One-way ANOVA 113 49 and a paired t-test were used to test the data for statistically sig- 114 50 nificant differences of agonist and antagonist compounds using SAS 115 51 9.3 (Cary, NC). Student-Newman-Keuls (SNK) post-hoc analysis was 116 52 used to determine significant differences between treatments with 117 53 95% confidence (a ¼ 0.05). Antagonist and modulator screening 118 54 results were normalized to 150 nM tyramine (100%), which is 119 55 represented as a dashed line in the appropriate figures. 120 56 121 57 3. Results 122 58 123

59 The putative tyramine receptor was successfully expressed in Fig. 1. Expression of RmTAR1 alone and in combination with G-protein chimeras. 124 60 Chinese Hamster Ovary (CHO) cells, and functional receptor acti- Transient expression of the R. microplus tyramine receptor coding sequence alone 125 a a a 61 vation was determined using a calcium-liberation assay. The (endogenous G q) and in combination with G-protein chimeras (G qs(5),Gqz(5), 126 Ga ,Ga ) in CHO cells. Intracellular calcium concentrations were measured with 62 calcium-liberation assay analyzes receptor activation via the Gaq qo(5) qi(5) 127 a calcium-sensitive fluorescent indicator (relative fluorescent units, RFU). Tyramine 63 phospholipase-C (PLC) pathway. Receptor activation results in an 128 and octopamine were screened at 1 mM to see which ligand strongly activated either fi 64 increase of cytosolic calcium, which can be quanti ed using a the receptor alone or the receptor in combination with specific G-protein chimeras. 129 65 calcium-sensitive fluorescent dye. However, not all receptors will Different letters indicate a statistically significant response (ANOVA, a ¼ 0.05). 130

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1 putative octopamine receptor (referred to here as the putative 66 2 tyramine receptor) aligns with the proliferation of tyramine re- 67 3 ceptor sequence data to show the receptor is a tyramine receptor. 68 4 Specifically, this receptor appears to be a tyramine-1 receptor 69 5 (RmTAR1) (Ohta and Ozoe, 2014). 70 6 A colonial cell line that stably expressed the cattle tick tyramine 71 7 receptor and the Gaqi(5) chimera was established and selected over 72 8 several weeks. Efficiency of the stable transfection of both the 73 9 tyramine receptor and the G-protein chimera was approximately 74 10 8% (n ¼ 24). Tyramine resulted in a concentration-dependent 75 11 response of CHO cells stably expressing the tyramine receptor 76 12 and the Gaqi(5) chimera, which had an EC50 of 15 nM. This is similar 77 13 to CHO cells transiently expressing the tyramine receptor and the 78 14 Gaqi(5) chimera (Fig. 4 and Table 1). 79 15 The pharmacological toolbox to characterize octopamine re- 80 16 ceptors and tyramine receptors is limited. Adrenergic agonists and 81 17 adrenergic antagonists are commonly used to determine the 82 18 pharmacology of octopamine receptors and tyramine receptors. Six 83 19 antagonists (propranolol, chlorpromazine, mianserin, phentol- 84 20 amine, cyproheptadine and yohimbine) were screened at three 85 21 concentrations (0.1 mM, 1 mM and 10 mM) to determine if they were 86 22 Fig. 2. Concentration-dependent response of transiently expressed RmTAR1 plus capable of antagonizing the receptor when the receptor was 87 a 23 G qi(5). Concentration-dependent response of the expressed coding sequence of the stimulated with 150 nM tyramine (Fig. 5). Propranolol enhanced 88 R. microplus tyramine receptor and the Ga G-protein chimera with several con- qi(5) fi 24 centrations of tyramine (solid black line) and octopamine (solid gray line). These two the effect of 50 nM tyramine, but it was not statistically signi cant 89 25 biogenic amines were used to monitor the increase of intracellular calcium, using a from tyramine alone (Fig. 5). Cyproheptadine and yohimbine were 90 26 calcium-sensitive fluorescent dye, measured by relative fluorescence units (RFU). CHO the only compounds capable of antagonizing 150 nM of tyramine 91 27 cells not transfected with the receptor were screened at various concentrations of significantly at all three concentrations (0.1 mM, 1 mM and 10 mM) 92 tyramine (dashed gray line). Data analyzed using GraphPad Prism ECanything (F ¼ 50 28 tested (Fig. 5). Mianserin antagonized 150 nM of tyramine at 1 mM 93 or EC50). 29 and 10 mM(Fig. 5). Phentolamine antagonized only at 10 mM. 94 30 Yohimbine and phentolamine both antagonized the 1 mM tyramine 95 31 Table 1 response in a concentration-dependent manner; however, yohim- 96 32 EC50 of tyramine and octopamine. This table shows the EC50 for the concentration- bine was 18-fold more effective at antagonizing the RmTAR1 97 33 dependent response of tyramine. The values were obtained when an R. microplus compared to phentolamine (Table 2). 98 tyramine receptor and the Gaqi(5) G-protein chimera were transiently and stably 34 transfected in CHO cells. Tyramine's activation of the receptor resulted in the in- Compounds that were able to stimulate or act as agonists to the 99 35 crease of intracellular calcium concentration, which was measured using a calcium- RmTAR1 were also examined. Agonists tested include clonidine, 100 36 sensitive fluorescent dye. naphazoline and tolazoline, which were screened at 0.1 mM, 1 mM 101 37 and 10 mM to see if they activated RmTAR1, compared to the same 102 EC50 ± SEM 95% CI 38 103 Tyramine (transient) 9 ± 0.9 nM (0.1 nM, 764 nM) 39 Octopamine (transient) 351 ± 2 nM (302 nM, 1476 nM) 104 40 Tyramine (stable) 15 ± 7 nM (5 nM, 60 nM) 105 41 106 42 107 43 108 44 109 45 110 46 111 47 112 48 113 49 114 50 115 51 116 52 117 53 118 54 119 55 120 56 121 57 122 58 123 Fig. 4. Tyramine concentration-dependent response of CHO cells transiently and sta- 59 124 bly expressing RmTAR1 plus Gaqi(5). Concentration-dependent response of the 60 expressed coding sequence of the R. microplus tyramine receptor and the Gaqi(5) G- 125 61 protein chimera. This concentration-dependent response compared CHO cells tran- 126 a 62 Fig. 3. Effect of biogenic amines against RmTAR1. Several biogenic amines were siently expressing the G qi(5) G-protein chimera (solid black), and CHO cells stably 127 a 63 examined to determine if they activated the expressed receptor and increased the expressing the R. microplus tyramine receptor with the G qi(5) G-protein chimera 128 intracellular concentration of calcium by using a calcium-sensitive fluorescent dye that (dashed gray). CHO cells not transfected with the receptor were screened at various 64 was measured in relative fluorescence units (RFU). Two-way ANOVA (a ¼ 0.05) concentrations of tyramine (dashed black line). Data analyzed using GraphPad Prism 129

65 different letters indicate a signiﬁcant difference. ECanything (F ¼ 50 or EC50). 130

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1 66 2 67 3 68 4 69 5 70 6 71 7 72 8 73 9 74 10 75 11 76 12 77 13 78 14 79 15 80 16 81 17 82 18 83 19 84 20 85 21 86 22 87 23 88 a 24 Fig. 6. Agonist screening against CHO cells stably expressing RmTAR1 and G qi(5) G- 89 protein chimera. Agonist screens were done in real time to determine if they were able 25 90 to increase the intracellular concentration of calcium, which was detected by a 26 calcium-sensitive fluorescent indicator. Agonist screening was performed at three 91 27 concentrations (0.1 mM, 1 mM, and 10 mM). An asterisk indicates a statistically signifi- 92 Fig. 5. Antagonist screening against CHO cells stably expressing RmTAR1 and Gaqi(5) 28 G-protein chimera. Antagonists were pre-incubated for 30 min prior to the addition of cant agonistic effect, compared to the respective concentration of tyramine. 93 29 tyramine, which was added at 150 nM. The effect of the antagonist was normalized to 94 30 this 150 nM tyramine response (100%, dashed line). Data was analyzed using a one- 95 a ¼ fi 31 way ANOVA ( 0.05). An asterisk indicates a statistically signi cant antagonistic 96 effect, compared to the addition of150 nM tyramine alone. 32 97 33 98 concentrations of tyramine (Fig. 6). Clonidine, naphazoline and 34 99 tolazoline did not significantly agonize RmTAR1 to the same level as 35 100 tyramine (Fig. 6). Naphazoline and tolazoline were able to partially 36 101 agonize RmTAR1 at 10 mM, just not as strongly as tyramine itself. 37 102 Clonidine, naphazoline and tolazoline were also screened as an- 38 103 tagonists or modulators of the tyramine receptor. This was per- 39 104 formed by preincubating these compounds for 30 min prior to the 40 105 addition of 150 nM tyramine. Tolazoline and naphazoline signifi- 41 106 cantly enhanced the 150 nM response of tyramine at all three 42 107 concentrations (0.1 mM, 1 mM and 10 mM), as shown in Fig. 7. 43 108 Previously, this putative tyramine receptor had been hypothe- 44 109 sized to be involved in resistance to the chemical acaricide amitraz, 45 110 which is a formamidine insecticide/acaricide. Amitraz's metabolite, 46 111 BTS-27271, was screened at 10 mM and 100 mM and significantly 47 112 increased the150 nM-tyramine response by 191 ± 16% and 48 113 150 ± 24%, respectively. This agonistic activity was only observed in 49 114 the presence of 150 nM tyramine (Fig. 8). 50 115 51 116 4. Discussion 52 117 53 118 Reintroduction of R. microplus into the United States would have 54 119 devastating consequences to the livestock industry. To aid in an 55 120 integrative chemical approach to controlling the southern cattle 56 121 57 122 58 Table 2 123 59 IC50 of yohimbine and phentolamine. This table shows the IC50 for the 124 concentration-dependent inhibition of receptor activation, normalized to 1 mM Fig. 7. Screening of agonists/modulators against RmTAR1. Agonist or modulators were 60 125 tyramine. The values were obtained with an R. microplus tyramine receptor and the preincubated for 30 min prior to the addition of 150 nM of tyramine (dashed line). The 61 Ga G-protein chimera that were stably transfected in CHO cells. response of the agonist or modulator was normalized to tyramines response (dashed 126 qi(5) a 62 line) Screening against CHO cells stably expressing RmTAR1 and G qi(5) G-protein 127 IC ± SEM 95% CI 63 50 chimera. Agonist screens were done in real time to determine if they were able to 128 increase the intracellular concentration of calcium. Screening was performed at three Yohimbine 280 ± 151 nM (73 nM, 1070 nM) 64 concentrations (0.1 mM, 1 mM, and 10 mM). An asterisk indicates a statistically signifi- 129 Phentolamine 5156 ± 969 nM (118 nM, 2.26 105 nM) 65 cant effect, compared to addition of 150 nM tyramine alone. 130

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1 a2-adrenergic antagonist, and has previously been shown to be 66 2 effective at blocking the inhibitory effect of tyramine on forskolin- 67 3 stimulated cAMP in heterologous expression systems (Ohta et al., 68 4 2003; Rotte et al., 2009; Wu et al., 2013). Rotte et al. (2009) 69 5 found that yohimbine produced the strongest antagonistic effect, 70 6 followed by chlorpromazine then cyproheptadine. However, we 71 7 find that yohimbine and cyproheptadine produce the strongest 72 8 antagonistic effect, at the three tested concentrations, followed by 73 9 mianserin (significant antagonism at 1 mM and 10 mM), then fol- 74 10 lowed by phentolamine (significant at 10 mM). Phentolamine is 75 11 widely accepted as a more efficient antagonist for octopamine re- 76 12 ceptors than for tyramine receptors (Roeder, 2005), which is 77 13 consistent with our results. 78 14 The tyramine receptor described here was also investigated for 79 15 its potential role in amitraz resistance observed in southern cattle 80 16 tick populations from Australia and the Americas (Baxter and Barker, 81 17 1999; Chen et al., 2007). The first identification of the tyramine re- 82 18 ceptor was in Australia, and no point mutations were discovered in 83 19 the amitraz-resistant strain compared to the amitraz-susceptible 84 20 strain (Baxter and Barker, 1999). However, the Santa Luiza and San 85 21 Alfonso strains of southern cattle tick display two point mutations 86 22 (T8P and L22S), and these ticks are resistant to amitraz (Chen et al., 87 23 2007). This was hypothesized to have a role in formamidine resis- 88 24 tance, and this is currently the topic of ongoing studies in our lab- 89 25 oratory. Recent studies have reinstated the Australian Rhipicephalus 90 26 tick as a separate species, Rhipicephalus (Boophilus) australis (Fuller, 91 27 1899) present in Australia, Southeast Asia and Indonesia (Estrada- 92

28 Pena et al., 2012, Barker et al., 2014; Burger et al. 2014). Separation of Q1,2 93 29 the Australian and American ticks into separate species suggests 94 30 potential differences in mechanisms for development of amitraz 95 31 resistance between the two species as it has been documented for 96 32 pyrethroid resistance (Guerrero et al., 2014). 97 a 33 Fig. 8. Amitraz metabolite's effect on RmTAR1 and G qi(5). The metabolite of amitraz Octopamine receptor(s) are believed to be the primary target of 98 a 34 (BTS-27271) was screened against CHO cells stably expressing RmTAR1 and G qi(5) G- formamidine metabolites, including amitraz's metabolite BTS- 99 protein chimera at two concentrations (10 mM and 100 mM). The metabolite was added 35 30 min prior to the addition of 150 nM tyramine. The response of the metabolite was 27271. Demethylchlordimeform (chlordimeform's metabolite) did 100 36 normalized to tyramines effect (dashed line). An asterisk indicates a statistically sig- not have an effect on forskolin-stimulated cAMP on B. mori's tyra- 101 37 nificant effect, compared to the addition of 150 nM tyramine alone. mine receptor when expressed in HEK-293 cells (Ozoe et al., 2004). 102 38 We also did not see any effect of amitraz's metabolite alone (data 103 39 tick, research is required to understand mechanisms of acaricide not shown); however, significant receptor activation was observed 104 40 resistance, and to identify new chemistries that either target when applied in combination with 150 nM tyramine (Fig. 7). This 105 41 known mechanisms of action or identify new mechanisms of toxic indicates that BTS-27271 can have an effect at the tyramine re- 106 42 action. The results shown here indicate that nucleic acid sequence ceptor but probably only when the tyramine receptor is activated. 107 43 originally thought to code for an octopamine receptor from Given the emerging evidence that populations around the world 108 44 R. microplus is actually a tyramine receptor. Additionally, functional may constitute a species complex, having an understanding of 109 45 assays showed that when this tyramine receptor was co-expressed amitraz-resistance may aid in adapting strategies to control of 110 a fi 46 with the G qi(5) chimera it resulted in the most signi cant response. southern cattle ticks. Recently, a mutation in a putative R. microplus 111 a a 47 The G qi(5) uses the G i receptor interacting domain; it is likely that b-adrenergic-like octopamine receptor has been hypothesized to 112 48 this tyramine receptor would signal down the pathway that has an have a role in amitraz-resistance (Corley et al., 2013). However, 113 49 inhibitory effect on adenylate cyclase, thereby, decreasing the functional receptor and functional resistance assays have not been 114 50 intracellular concentration of cAMP. conducted against this octopamine receptor to date. 115 51 Recent reviews have hypothesized, based on sequence data, that 116 52 this receptor is a TAR1 (this class was previously known as octop- Uncited reference 117 53 amine/tyramine or tyramine receptor), which would normally 118 a 54 signal through the G i pathway (Ohta and Ozoe, 2014; Verlinden Huang et al., 2009. Q4 119 55 et al., 2010; Farooqui, 2012). Several TAR1 receptors have been 120 56 identified and second messenger pathways analyzed from Apis Acknowledgment and Disclaimer 121 57 mellifera, AJ245824 (Blenau et al., 2000), Bombyx mori, AB162828 122 58 (Ohta et al., 2003), Chilo Suppressalis, JQ416145 (Wu et al., 2013), This publication was developed under STAR Fellowship Assis- 123 59 Drosophila melanogaster, X54794 (Saudou et al., 1990), Heliothis tance Agreement no. FP917332 awarded by the U.S. Environmental 124 60 virescens, X95606 (von Nickisch-Rosenegk et al., 1996), Locusta Protection Agency (EPA). It has not been formally reviewed by the 125 61 migratoria, X69520 (Vanden Broeck et al., 1995) and Periplaneta EPA. The views expressed in this publication are solely those of the 126 62 americana, AM990461 (Rotte et al., 2009). authors, and EPA does not endorse any products or commercial 127 63 Pharmacological characterization of tyramine receptors, like services mentioned in this publication. USDA is an equal opportu- 128 64 octopamine receptors, is performed with agonists and antagonists nity provider and employer. This is a publication of the Iowa 129 65 used for the mammalian adrenergic system. Yohimbine is a known Agriculture Experiment Station. 130

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