Generation of Full-Length cDNAs for Eight Putative GPCnR from the Cattle Tick, R. microplus Using a Targeted Degenerate PCR and Sequencing Strategy

We describe here a rapid and efficient method for the targeted isolation of specific members of gene families without the need for cloning. Using this strategy we isolated full length cDNAs for eight putative G-protein coupled neurotransmitter receptors (GPCnR) from the cattle tick Rhipicephalus (Boophilus) microplus. Gene specific degenerate primers were designed using aligned amino acid sequences of similar receptor types from several insect and arachnid species. These primers were used to amplify and sequence a section of the target gene. Rapid amplification of cDNA ends (RACE) PCR was used to generate full length cDNA sequences. Phylogenetic analysis placed 7 of these sequences into Class A G-protein coupled receptors (GPCR) (Rm_α2AOR, Rm_β2AOR, Rm_Dop1R, Rm_Dop2R, Rm_INDR, Rm_5-HT7R and Rm_mAchR), and one into Class C GPCR (Rm_GABABR). Of the 7 Class A sequences, only Rm_mAchR is not a member of the biogenic amine receptor family. The isolation of these putative receptor sequences provides an opportunity to gain an understanding of acaricide resistance mechanisms such as amitraz resistance and might suggest possibilities for the development of new acaricides.


Introduction
The isolation of specific members of gene families typically involves the use of degenerate primers designed in regions conserved across many members of the gene family of interest. The subsequent PCR product requires cloning, as it contains amplicons from multiple genes. Clones are then sequenced looking for the gene of interest. This strategy is labour intensive, time consuming and often fails to isolate the target gene. We describe here a rapid, targeted approach which enables isolation of the gene of interest without cloning, using a degenerate PCR and sequencing strategy based on homologous amino acid motifs specific to each target gene.
GPCR are characterised by 7 trans-membrane spanning domains (TM) that contain the ligand-binding site, an extracellular amino-terminus, and an intracellular carboxyl-tail [1]. Ligand binding to the GPCR causes interactions with G proteins, mediating a series of intracellular functional responses via the second messengers, adenyl cyclase and or phospholipase C [2]. Between one third [3] and a half [4] of currently marketed human drugs, target GPCR. The cattle tick, Rhipicephalus (Boophilus) microplus is a very important parasite of cattle throughout the world and its control relies heavily on acaricides. The pyrethroid, formamidine and macrocyclic lactone families of acaricides are among the most widely used acaricides for controlling cattle ticks at present; all of which are known or believed to target membraneassociated proteins, potentially including GPCR. To date only two G-protein coupled neurotransmitter receptors have been described in the cattle tick [5,6]. We believe that R.microplus GPCR involved in the regulation of vital physiological functions offer valuable targets for new acaricides. Here we report the targeted isolation of eight putative G-protein coupled neurotransmitter receptors from the cattle tick, R. microplus.

Results
Using the gene specific degenerate primers designed with CODEHOP software (Figures 1, 2, 3), fragments from the eight target genes were amplified from both genomic (gDNA) and cDNA. The amplicons ranged in size from 234-1277 bp ( Figure 4) and were the same size for both gDNA and cDNA indicating that each degenerate primer pair was contained within one exon. Sequencing with degenerate primers yielded a single clean sequence from each amplicon. Following RACE PCR eight complete coding sequences were generated. Through phylogenetic analysis, these sequences could be grouped into two distinct classes of GPCR; Class A GPCR (or Rhodopsin like GPCR) and Class C GPCR. Figure 5 shows a phylogenetic diagram of insect and arachnid GPCR, including the 8 putative receptors reported in this paper. These receptors have been submitted to GenBank under the following accession numbers. Class A) Rm_a2AOR (JN974908), Rm_b2AOR (JN974909), Rm_Dop1R (JN974914), Rm_Dop2R (JN974912), Rm_INDR (JN974911), Rm_5-HT 7 R (JN974910) and Rm_mAchR (JN974913). Class C) Rm_GABA B R (JN974907).

Class A GPCR
Seven of the 8 sequences were grouped as Class A GPCR. These were the putative octopamine, dopamine, 5-hydroxytryptamine (5-HT 7 ) and muscarinic acetylcholine (mAch) receptors. As well as the 7 trans-membrane (TM) spanning domains ( Figure S1) typical of all GPCR, Class A receptors share a number of conserved features. There is an Asp-Arg-Tyr (DRY) or Asp-Arg-Phe (DRF) motif located in the second intracellular loop. This motif is important for the conformational changes involved in receptor activation. It is highly conserved among the Class A GPCR; sometimes this motif is also present as Glu-Arg-His (ERH) [7] or as Glu-Arg-Tyr (ERY) [8] but in all cases, within this class, the Arg residue is conserved. This motif is believed to be located in a hydrophilic pocket formed by polar residues from TM I, TM II and TM VII. Agonist binding causes protonation of the aspartic or glutamic acid residue, causing the Arg residue to move out of the pocket [9]. Two cysteines, one present in the first extracellular loop and the second in the second extracellular loop, are expected to form a disulphide bond which stabilizes the receptor [10,11]. All these features are present in the 7 sequences grouped in Class A ( Figures S2 & S3).
Biogenic Amine GPCR. Six of the 7 Class A receptors belong to the biogenic amine receptor family. These are the putative octopamine, dopamine and 5-HT 7 receptors. In addition  Table S1. doi:10.1371/journal.pone.0032480.g002 to features common to all Class A GPCR, characteristics typical of biogenic amine receptors include the presence of an aspartate (D) in trans-membrane domain TM III. This acts as a counter-ion for binding of the amine group. Two serines in TM V form hydrogen bonds with the catechol hydroxyl groups, and a phenylalanine in TM VI interacts with the catechol aromatic ring [12]. An aspartate residue in TM II which is involved in receptor activation is also conserved ( Figures S3 & S4). These features are present in the 6 putative biogenic amine receptors with the exception of the 5-HT 7 receptor, in which the two serines in TM V are replaced by two alanines. The other 5-HT receptor (5-HT 1 like) previously isolated from R. microplus [6] contain a serine and alanine at these positions ( Figure S4).
Putative Octopamine Receptors. Two of the newly generated sequences were phylogenetically most similar to octopamine receptors, which have been proposed as the target of amitraz [13,14].This class of receptor is preferentially activated by octopamine. Octopamine is a major neurotransmitter, neuromodulator and neurohormone, regulating diverse physiological processes in invertebrates, including fight or flight responses, egg-laying, sensory information processing, and complex neural functions such as learning and memory [15]. Sequence JN974908 (Rm_a2AOR ) is most similar to a-adrenergic-like octopamine receptors (aAOR). These receptors show the greatest similarity structurally and pharmacologically to vertebrate a-adrenergic receptors. They mediate their effects via increases in intracellular calcium levels [16]. Sequence JN974909 (Rm_b2AOR) is most similar to b-adrenergic-like octopamine receptors (bAOR). This class of receptors shows the greatest similarity structurally and pharmacologically to vertebrate badrenergic receptors [17]. In the presence of octopamine, increases in intracellular cAMP levels occur.
Putative Dopamine Receptors. Three of the newly generated sequences were phylogenetically most similar to invertebrate dopamine receptors. These receptors are preferentially activated by dopamine. Dopamine has been demonstrated to activate flight motor activity in Manduca sexta [18]. Dopamine has also been shown to reduce the response to conditioned stimuli as well as inhibiting retrieval of learned information in Apis mellifera [19]. Sequence JN974914 (Rm_Dop1R) is most similar to dopamine type 1 receptors (Dop1R). This class is most closely related to vertebrate D1 receptors and increases intracellular cAMP levels in the presence of dopamine. Sequence JN974912 (Rm_Dop2R) is most similar to dopamine type 2 receptors (Dop2R). These receptors share the closest homology with vertebrate D2 receptors and treatment with dopamine decreases intracellular cAMP. Sequence JN974911 (Rm_INDR) is most similar to invertebrate dopamine receptors (INDR). Although, as with D1 receptors, stimulation with dopamine increases intracellular cAMP levels [20], these receptors are more closely related structurally to invertebrate octopamine receptors.
Putative 5-HT 7 Receptor. Sequence JN974910 (Rm_5-HT 7 R) is most closely related to invertebrate 5-HT 7 receptors that mediate their effects by increasing intracellular cAMP levels. 5-HT has been demonstrated to enhance circadian rhythmdependent general motor activity in the moth Lymantria dispar, while suppressing dopamine-induced flight motor activity in Manduca sexta [18]. It has also been shown to reduce conditioned olfactory responses in the honeybee Apis mellifera [19,21].
Putative Muscarinic Acetylcholine Receptor (mAchR). Sequence JN974913 (Rm_mAchR) is most closely related to invertebrate muscarinic acetylcholine receptors (mAchR). Agonist binding of these receptors typically decreases intracellular cAMP levels by inhibiting adenylate cyclase or stimulating phospholipase C and the turnover of inositol phosphates. Muscarinic agonists have been shown to be effective acaricides [22,23]. Muscarinic receptors have been credited with two main functions in insects: inhibition of transmitter release from sensory neuron terminals and regulation of the excitability of motoneurons and interneurons [24].

Class C GPCR
Putative Metabotropic c-Aminobutyric Acid Receptor (GABA B R). A single sequence grouped with Class C GPCR. This was the putative metabotropic GABA B (c-aminobutyric acid) receptor JN974907 (Rm_GABA B R). Features characteristic of Class C GPCR are an N-terminal signal peptide, followed by a region with high sequence similarity to bacterial periplasmic amino acid binding proteins [25]. This constitutes the ligand binding site, located in a large extracellular N-terminal domain. The intracellular carboxy terminus is exceptionally large in all GABA B receptors. This protein segment contains a coiled-coil domain, which was shown to be necessary for the formation of GABA B R heterodimers in mammalian receptors [26,27]. The putative GABA B receptor sequence generated from R. microplus contains these features ( Figure S5). GABA (c-aminobutyric acid) functions as the primary inhibitory neurotransmitter in the central nervous system of vertebrates and invertebrates [28]. Stimulation with GABA causes a reduction in intracellular cAMP enabling regulation of K + and Ca 2+ . Ionotropic GABA A receptors are distributed throughout both the central and peripheral insect nervous systems [29] and are important targets for insecticides. Much less work has been done with metabotropic GABA B receptors and they too may offer important insecticide targets.

Discussion
Next generation sequencing technologies have seen dramatic decreases in cost and time associated with whole genome sequencing. This has seen the increased use of bioinformatics approaches for gene isolation. Despite this, degenerate PCR remains a powerful tool for gene isolation. The technique of designing degenerate primers in regions conserved across many members of the gene family of interest can be likened to a ''shotgun'' approach where amplicons from many genes are generated in the hope that the gene of interest is among these. Cloning is used to determine if the target gene has been amplified. This ''hit or miss'' strategy is both labour intensive, time consuming and ultimately may not generate the desired result .We report here on a targeted degenerate PCR and sequencing strategy for gene isolation. This strategy avoids amino acid motifs highly conserved across different gene types when designing degenerate primers and concentrates on motifs conserved only within the same gene type. This technique can be used with both gDNA and cDNA. By using a product such as GenomeWalker TM (Clontech Laboratories Inc. Mountain View, USA) to replace RACE PCR, genes can also be isolated from gDNA enabling gene isolation from samples unsuitable for RNA extraction. Using this process we were able to target and isolate eight putative  Table S2. The radial phylogram was constructed using Dendroscope software [33]. Dopamine receptor nomenclature is as proposed by Mustard et al., (2005). Octopamine receptor nomenclature is as proposed by Evans et al., (2005). Previously undescribed putative R. microplus receptors are in larger blue type. doi:10.1371/journal.pone.0032480.g005 Rhipicephalus (Boophilus) microplus G-protein coupled neurotransmitter receptor sequences without the need for cloning.
In other organisms, these GPCR have been shown to be involved in mediating a wide and diverse range of physiological processes. The ability to disrupt or alter these processes forms the basis by which many insecticides and acaricides act. The formamidine acaricide amitraz is believed to target the octopamine receptor, while muscarinic agonists have been shown to be effective acaricides. Ionotropic GABA A receptors are important targets for insecticides while only limited information about metabotropic GABA B receptors is available. 5-HT and dopamine receptors are also believed to mediate important physiological processes and may offer targets for new acaricides. An important consideration when developing new acaricides are their possible toxic effects on non-target organisms. The isolation of these putative receptor sequences will allow the expression of these receptors and the subsequent screening of agonists and antagonists. The identification of taxa-or species-specific ligands will aid in the development of more specific and safer acaricides. Combined with the opportunity to gain an understanding of acaricide resistance mechanisms, the development of new acaricides is important for the continued control of the cattle tick, Rhipicephalus (Boophilus) microplus, responsible for an estimated US$ 2 billion in annual economic losses worldwide [30].

cDNA Synthesis
Total RNA was purified from unfed tick larvae (Non Resistant Field Strain (NRFS)) [31] maintained at Biosecurity Science Laboratories (BSL) of the Department of Employment, Economic Development and Innovation (DEEDI) in Brisbane, Queensland. Approximately one gram of tick larvae was crushed under liquid nitrogen using a mortar and pestle. RNA extraction was performed using the TRIzolH Reagent (Invitrogen Life Technologies, Carlsbad, USA) following the manufacturer's protocol. Poly-A RNA was purified from total RNA using a POLY(A)Purist TM kit (Applied Biosystems/Ambion, Austin, USA). First strand cDNA synthesis was carried out using a Clontech SMARTer TM RACE cDNA Amplification Kit (Clontech Laboratories Inc. Mountain View, USA) following the manufacturer's directions.

PCR and sequencing with degenerate primers
Amino acid sequences (Table S1) were aligned for each receptor of interest using ClustalW2 software (European Bioinformatics Institute, Cambridge, UK). Degenerate primers were designed ( Figure 1) using CODEHOP software [32] from the aligned amino acid sequences. Each CODEHOP degenerate primer consisted of a pool of related primers containing all possible nucleotide sequences encoding 3-4 highly conserved amino acids within a 39 degenerate tail. A longer 59 non-degenerate clamp region contained the most probable nucleotides predicted for each flanking codon. The most probable nucleotides in the 59 clamp for Rhipicephalus microplus were predicted from codon usage tables in CODEHOP. Wherever possible, primers chosen were specific to the receptor of interest only. Primer specificity was determined using the 39 degenerate tail. (Figures 2 & 3) To determine the suitability for use with different templates, degenerate PCR was carried out on both gDNA and cDNA. If gDNA is the primary source of template, primers should be designed to amplify small fragments of less than 200 bp to increase the chance of both priming sites being contained within the same exon. 30 ng of template was amplified in a 25 ml reaction volume containing; 1 ml degenerate primers (primers were prepared at a concentration equal to 10 mM6level of degeneracy), 2 ml cDNA, 16 KAPA2G PCR Buffer B, 16 KAPA PCR Enhancer 1, 1.5 mM MgCl 2 , 0.2 mM dNTP and 0.05 U/ml KAPA2G Robust DNA Polymerase (KAPABiosystems. Woburn, USA). PCR conditions were: initial denaturation 95uC for 5 min, followed by 8 cycles of 94uC for 15 s, 63uC for 30 s decreasing by 1uC per cycle, 72uC for 2 min, followed by 27 cycles of 94uC for 15 s, 56uC for 30 s, 72uC for 2 min, followed by a final extension of 72uC for 7 min. PCR products were separated on a 1.5% agarose gel (Figure 4). Products of the correct size were sampled with a 10 ml pipette tip. This was used as template and re-amplified as above except with degenerate primers at a concentration of 10 mM. Unused dNTPs and primers were removed from the PCR product using Exosap-itH (USB Corporation distributed by GE Healthcare Bio-Sciences, Rydalmere, Australia). Sequencing was performed using an ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit Version 3.1 (PE Applied Biosystems, Foster City, USA). The sequencing reaction contained 30 ng template DNA, 1 ml degenerate PCR primer (5 mM), 4.5 ml of 56 sequencing buffer, 1 ml Big Dye Terminator, to a final volume of 20 ml with MilliQ H 2 O. In a single case an internal degenerate sequencing primer was designed as both of the degenerate PCR primers failed to give clean sequence. This was used at a concentration equal to 5 mM6level of degeneracy. Sequencing separation was performed on an ABI 3130xl automated sequencer. Forward and reverse sequences were aligned and edited using ChromasPro (Technelysium Pty Ltd, Tewantin, Australia).

RACE PCR
5RACE and 3RACE ready cDNA was prepared using a Clontech SMARTer TM RACE cDNA Amplification Kit following the manufacturer's directions. Using the sequences generated by degenerate PCR in the previous section and following the manufacturer's instructions, 39 and 59 gene specific RACE primers were designed and RACE PCR was performed. PCR products were separated on a 1.5% agarose gel. Products of the correct size were sampled with a 10 ml pipette tip and re-amplified. PCR products were cleaned and sequenced as described above. Internal sequencing primers were designed, where necessary, to enable sequencing of full length cDNAs. sapiens_GABA B receptor: CAA09940, Rm_GABA B R _Rhipicephalus (Boophilus) microplus_GABA B receptor: JN974907 . Signal peptide sequences were predicted using SignalP 3.0. [35] Coiledcoil domains were predicted using COILS [36]. (DOC) Table S1 Accession numbers for amino acid and nucleic acid sequences used for designing degenerate primers.