Adult R. prolixus were maintained with a 12 h light/dark cycle reared at ~50% humidity and 28°C and fed defibrinated rabbit's blood (Hemostat Laboratories, Dixon, CA, USA; supplied by Cedarlane Laboratories Inc., Burlington, ON, Canada) once in every instar.

All biogenic amines (D, L-octopamine hydrochloride, tyramine hydrochloride, serotonin hydrochloride and dopamine hydrochloride) were made as 10⁻² M stocks dissolved in double distilled water. All biogenic amine antagonists (phentolamine hydrochloride, gramine, metoclopramide hydrochloride, mianserin hydrochloride, cyproheptadine hydrochloride, phenoxybenzamine hydrochloride, yohimbine hydrochloride, and chlorpromazine hydrochloride) were prepared in molecular grade ethanol or dimethyl sulfoxide to 10⁻² M stocks. The final percentage of solvent in the experimental treatments was ≤0.1%. All chemicals were obtained from Sigma Aldrich (Oakville, Canada).

The scaffold, transcripts and proteins of R. prolixus were uploaded into Geneious 8.1 (Auckland, New Zealand) from vectorbase.org. Using D. melanogaster Octβ2-R-PA (Q4LBB9) and D. melanogaster Tyr1-R (Q9VEG1) as templates, a tblastn search against R. prolixus transcripts and proteins as performed. The partial cDNA sequences of the closest results to the query sequence obtained for Octβ2-R (RPRC011545) and Tyr1-R (RPRC008712) were amplified by specific primers for each receptor (Table S1). OneTaq® DNA Polymerase (NEB, Whitby, ON, Canada) was used for all PCRs. The cycling profiles for the PCRs using Bio-Rad's s100 thermocycler (Bio-Rad Laboratories, Mississauga, ON, Canada) were: initial denaturation at 94°C for 5 min, followed by 29 cycles at 94°C for 30 sec, 55–63°C annealing for 1 min, 68°C for 1 min and a final extension at 68°C for 5 min. Products from the reactions were gel extracted using EZ-10 Spin Column DNA Gel Extraction Kit (Bio Basic, Markham, ON, Canada) and cloned using pGEM-T Easy Vector (Promega, Madison, WI, USA). White colonies containing the inserts, tested using PCR, were inoculated and left overnight to grow. The inserts were extracted using EZ-10 Spin Column Plasmid DNA MiniPreps Kit (Bio Basic, Markham, ON, Canada) and sent for Sanger sequencing at the Centre of Applied Genomics at the Hospital for Sick Children (Toronto, ON, Canada) or Eurofins Genomics (Toronto, ON, Canada).

Gene specific forward primers (Table S2) were used along with pDNR-LIB−25 Revs plasmid primer to amplify the cDNA regions at the 3′ end of the receptors (3′ Modified RACE). The product of the first reaction was nested with another gene specific primer (Table S2). This process was repeated until one characteristic band for each receptor was observed. The bands were gel purified, cloned and sequenced. Many gene specific forward primers (see Table S3) were designed for the amplification of the 5′ cDNA ends of both receptors (5′ Modified RACE). Essentially, a series of nested PCRs were utilized to distinguish the correct bands. The products of the first reaction were purified and used as a template for the subsequent reaction. Selected fragments were gel extracted, cloned and sequenced. The cDNA sequences for RhoprOctβ2-R (MF377526) and RhoprTyr1-R (MF377527) have been deposited in GenBank.

The seven transmembrane domains for both receptors were predicted by TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). BLAST was used to predict exon-intron boundaries for both receptors, this was confirmed by a fruit fly splice site prediction tool (http://www.fruitfly.org/seq_tools/splice.html). The intracellular and N-glycosylation sites were predicted using NeyGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) and the intracellular phosphorylation sites were predicted using NetPhos 3.1 Server (http://www.cbs.dtu.dk/services/NetPhos/). Palmitoylation of cysteine residues were predicted by GPS.Lipid. An Integrated Resource for Lipid Modifications predictor (http://lipid.biocuckoo.org/webserver.php).

The open reading frames (ORF) of both RhoprOctβ2-R and RhoprTyr1-R were amplified using Q5® High-Fidelity DNA Polymerase (New England Biolabs, Massachusetts, United States) and the Kozak translation initiation sequence (GCCACC) was inserted at the 5′ end of each receptor (Table S4; Kozak, 1987). The products were cloned into a pGEM-T Easy vector (Promega, Madison, WI, USA) and sequenced. The receptors were reamplified with Bgl II restriction site introduced at the 5′ end and Bam HI site introduced at the 3′ end of each receptor (Table S4). RhoprOctβ2-R was subcloned into pIRES2-ZsGreen1 (Clontech, Mountain View, CA, USA) and RhoprTyr1-R was subcloned into pIRES2 DsRed-Express2 (Clontech, Mountain View, CA, USA).

A HEK293/CNG cell line that stably expresses a modified cyclic nucleotide-gated channel (CNG) (previously available from BD Biosciences, Mississauga, ON, Canada) were raised in Dulbecco's Modified Eagle Medium Nutrient Mixture F12-Ham (DMEM/F-12) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin and streptomycin, and 100 μg/mL G418. The cells were incubated at 37°C in 5% CO₂. The cells were grown in T75 flasks to 90–95% confluency and were transiently co-transfected with either expression vector containing the receptor and aequorin at a 2:1 ratio (transfection reagent to expression vectors) using X-tremeGENE® HP DNA Transfection Reagent (Roche Applied Science, Penzberg, Germany). The cells were incubated for 72 h and used for the functional cell assay.

HEK293/CNG cells were harvested with PBS-EDTA solution and placed in working bovine serum albumen (BSA) medium (DMEM/F-12 with 1% BSA and 1% penicillin and streptomycin). The cells were incubated in coelenterazine h (Promega, Madison, WI, USA) to a final concentration of 5 μM with stirring in the dark for at least 3 h. Before running the bioluminescence assay, the cells were diluted by 5-fold using the BSA medium. For the dose-response curves, stock solutions of D, L-octopamine hydrochloride and tyramine hydrochloride were diluted in BSA medium and tested in triplicate in flat bottom CELLSTAR 96 well-plates (Greiner Bio-One, Kremsmunster, Austria). Using an automated injector, 50 μL of cells were loaded into each well and luminescence was measured over three intervals for 15 s using a Wallac Victor2 plate reader (Perkin Elmer, San Diego, CA, USA). The agonists, serotonin and dopamine, were tested in a similar manner as above. For the antagonists assay, octopamine or tyramine were loaded into the wells along with each antagonist, all dissolved in BSA medium. The plate was then vortexed for 2 min before running the assay.

The expression of both receptors was examined in 1 week old, unfed female, 4 weeks post-feeding as fifth instars. The central nervous system (CNS), ovary (OVA), lateral oviducts (LOV), common oviduct and spermatheca (COS), bursa (BUR), cement (CEM) were dissected and placed in nuclease-free phosphate-buffered saline (PBS) (Sigma Aldrich, Oakville, ON, Canada). Total RNA extraction was followed using EZ-10 Spin Column Total RNA Minipreps Super kit (Bio Basic, Markham, ON, Canada) and cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Mississauga, ON, Canada). The total cDNA produced was diluted by 10-fold and used for the quantitative PCR reactions. The RhoprOctβ2-R, RhoprTyr1-R and the reference genes (α-tubulin, β-actin, ribosomal protein 49) were amplified by Mx35005 Quantitative PCR System (Stratagene, Mississauga, ON, Canada) using the primers provided (Table S5) and SsoFAST EvaGreen Supermix with low Rox (Bio-Rad, Mississauga, ON, Canada). The reaction conditions for both receptors started with an initial denaturation at 95°C for 30 sec followed by 40 cycles of denaturation at 95°C for 5 s, annealing and extension at 60°C for 33 s. Two technical replicates were performed per tissue along with a non-template control for each biological replicate. Analysis of relative expression levels was determined using the ΔCt method. Normalized expression was obtained by first averaging the C_T values of the reference genes β-actin, α-tubulin and rp49; and second comparing these values to the C_T values of the gene of interest in all tissues.

GraphPad Prism version 5.03 (www.graphpad.com) was used to create and statistically analyze all graphs in this paper.

Various potential biogenic amine receptors were analyzed in silico using D. melanogaster receptors as templates. Two targets were selected for 5′ and 3′ amplification by PCR. The full sequences of RhoprOctβ2-R and RhoprTyr1-R were obtained by a modified method of Rapid Amplification of cDNA Ends (RACE). The total length of the RhoprOctβ2-R (MF377526) cDNA sequence amplified was 1,799 bp yielding 447 amino acids with a molecular weight of 50,431 kDa (Figure 2A). Regions within the arrows indicate the predicted cDNA regions available on VectorBase (Figure 2A). The ORF of RhoprOctβ2-R spanned four exons with lengths of 455, 286, 353, and 599 bp separated by three intronic regions (Figure 2B). The length of the RhoprTyr1-R (MF377527) cDNA sequence amplified was 1,496 bp resulting in 455 amino acids with a molecular weight of 51,925 kDa (Figure 3A). The cDNA region within the first arrow and the second arrow + the third and the fourth arrow indicate predicted sequences obtained from VectorBase (Figure 3A). The RhoprTyr1-R ORF spanned a single exon with a length of 1,526 bp (Figure 3B). Furthermore, both receptors belong to the rhodopsin-like (Class A) GPCR superfamily characterized by seven transmembrane hydrophobic domains (TM), a DRY residue in the TM3 and an NPxxY motif in TM7 (Rovati et al., 2007; Rosenbaum et al., 2009). The amino terminal of RhoprOctβ2-R consisted of 65 amino acids compared to 49 amino acids for the RhoprTyr1-R (Figures 2, 3). The carboxyl terminal of RhoprOctβ2-R consisted of 66 amino acids vs. only 18 amino acids for RhoprTyr1-R. Characteristic to all Tyr1-Rs, the third intracellular loop of RhoprTyr1-R (151 amino acids) is elongated when compared to RhoprOctβ2-R's (70 amino acids) third intracellular loop (Figures 2A, 3A). In conclusion, the cloned cDNA sequences encode amino acid sequences predicted to form two GPCRs, RhoprOctβ2-R and RhoprTyr1-R. These newly found GPCRs contain all the structural features predicted to be required for function.

In order to determine the correct designation of the two receptors cloned, phylogenetic analysis was conducted with various insect octopamine and tyramine receptors. Different receptor types form separate monophyletic groups (Figure 4). Octβ-Rs form a large monophyletic group with each subtype of Octβ-Rs forming a separate monophyletic group. The Octα-Rs and the Tyr1-Rs form the other major monophyletic group. The single D. melanogaster Tyr3-R is part of Tyr2-R monophyletic group (Figure 4). RhoprOctβ2-R shared 78% identity and 87% similarity with N. lugens's Octβ2-R (ASA47149.1). RhoprTyr1-R shared 56% identity and 64% similarity with Tyr1-R of D. melanogaster (NP_001163494.1). Now that the correct designations have been established for both receptors, it is essential to consider the predicted biochemical features of these GPCRs. Post-translational modifications such as phosphorylation, N-glycosylation and cysteine palmitoylation of the receptors are essential for the structural integrity and functionality of the receptors (Kristiansen, 2004). RhoprOctβ2-R is predicted to be N-glycosylated at two sites at Asn⁴⁰ and Asn⁴³ (Figure 2A). Multiple serine and threonine residues are predicted to be phosphorylated by kinases in the third intracellular loop and the C-terminus (Figure 2A). For RhoprTyr1-R, N-glycosylation is noted at two sites, Asn¹⁴ and Asn¹⁷, at the N-terminus (Figure 3A). Multiple phosphorylation sites are highlighted in the long third intracellular loop between TM5 and TM6 (Figure 3A). Multiple sequence alignment of octopamine and tyramine receptors yielded key amino acids common in all insect octopamine and tyramine receptors (Figures 5, 6). In short, RhoprOctβ2-R and RhoprTyr1-R contain the predicted biochemical sites suggesting that these receptors are functionally viable and are closely related to other insect Octβ2 and Tyr1 receptors.

It was important to confirm the identity of RhoprOctβ2-R and RhoprTyr1-R by testing the activation of the receptors to their corresponding ligands. This was done by transfecting and transiently expressing both receptors in HEK293/CNG cells. The interaction of the ligand with the receptor was monitored by measuring the bioluminescence released due to calcium mobilization in the cytosol. Maximum activation of the receptors, peak luminescence, was detected within 5–10 s. RhoprOctβ2-R was activated in a dose-dependent manner by both octopamine and tyramine (Figure 7A). Tyramine (EC₅₀ = 3.85 × 10⁻⁶ M) was ten times less potent than octopamine (EC₅₀ = 3.67 × 10⁻⁷ M) in activating RhoprOctβ2-R. Tyramine was a partial ligand of RhoprOctβ2-R with a 69.00 ± 7.94% luminescence response relative to 10⁻⁵ M octopamine (One-Way ANOVA followed by Dunnett's Multiple Comparison Test compared to the octopamine group at 100%, ^**P < 0.01) (Figure 7B). Other biogenic amines (serotonin and dopamine) were inactive against RhoprOctβ2-R, confirming its selectivity for octopamine and tyramine (Figure 7B). For the antagonists, phentolamine and gramine significantly reduced the 10⁻⁵ M octopamine-induced luminescence response, whereas partial inhibition was noted with chlorpromazine, mianserin and metoclopramide (One-Way ANOVA followed by Dunnett's Multiple Comparison Test compared to the octopamine group at 100%, ^*P < 0.05, ^***P < 0.001) (Figure 7C). Furthermore, tyramine and octopamine each activated RhoprTyr1-R in a dose-dependent manner with a threshold in the nanomolar range for tyramine (Figure 8A). Tyramine (EC₅₀ = 5.17 × 10⁻⁸ M) was ~100 times more potent that octopamine (EC₅₀ = 6.88 × 10⁻⁶ M). Analysis of various biogenic amine agonists (serotonin and dopamine) against RhoprTyr1-R revealed that RhoprTyr1-R is selective for tyramine and octopamine (yields ~50% of 10⁻⁵ M tyramine luminescence response) (Figure 8B). Various biogenic amine antagonists were effective in significantly reducing tyramine's luminescence response, most notably, yohimbine (>50% reduction in luminescence) and phenoxybenzamine (One-Way ANOVA followed by Dunnett's Multiple Comparison Test compared to the tyramine group at 100%, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001) (Figure 8C). HEK293/CNG cells transfected with empty vectors resulted in a luminescence response that was identical to control wells. As predicted, RhoprOctβ2-R and RhoprTyr1-R are functional GPCRs. Octopamine and tyramine bind to RhoprOctβ2-R and RhoprTyr1-R causing modification in second messengers.

The expression of both receptors was analyzed in the CNS and in the reproductive system of adult female R. prolixus using Real Time Quantitative PCR. RhoprOctβ2-R transcript expression was highly expressed in the CNS relative to the female adult reproductive tissues (Figure 9A). RhoprOctβ2-R expression was roughly similar in all reproductive tissues (Figure 9A). Slightly low expression of RhoprOctβ2-R transcript was found in the bursa compared to other reproductive tissues (Figure 9A). Transcript distribution of RhoprTyr1-R was enriched in the CNS relative to adult female reproductive tissues (Figure 8B). The expression of RhoprTyr1-R in the ovary and common oviduct + spermetheca was similar (Figure 9B). Low RhoprTyr1-R expression was noted in the lateral oviducts and the cement gland (Figure 9B). Overall, the expression of RhoprOctβ2-R and RhoprTyr1-R in the reproductive system suggests that octopamine and tyramine could utilizing these receptors to cause a modification in the rhythmic contractions of the reproductive visceral muscle (Hana and Lange, 2017). In fact, tyramine's lack of direct action at the oviducts could be substantiated by the lower expression of RhoprTyr1-R transcript relative to other tissues (Hana and Lange, 2017).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.