Characterization of a tyramine receptor type 2 from hemocytes of rice stem borer, Chilo suppressalis

Journal of Insect Physiology 75 (2015) 39–46

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Characterization of a tyramine receptor type 2 from hemocytes of rice stem borer, Chilo suppressalis Shun-Fan Wu a,b,⇑, Gang Xu b, Gong-Yin Ye b,⇑ a b

State & Local Joint Engineering Research Center of Green Pesticide Invention and Application, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China State Key Laboratory of Rice Biology & Key Laboratory of Agricultural Entomology of Ministry of Agriculture, Institute of Insect Sciences, Zhejiang University, Hangzhou, China

a r t i c l e

i n f o

Article history: Received 21 January 2015 Received in revised form 1 March 2015 Accepted 6 March 2015 Available online 12 March 2015 Keywords: G protein-coupled receptor Hemocytes Octopamine Tyramine Chilo suppressalis

a b s t r a c t Calcium acts as a second messenger in many cell types, including insect hemocytes. Intracellular calcium level has a definite role in innate and adaptive immune signaling. Biogenic amines such as octopamine (OA), tyramine (TA), dopamine (DA) and serotonin (5-HT) play various important physiological roles in insects by activating distinct G-protein-coupled receptors (GPCRs) that share a putative seven transmembrane domain structure. OA and 5-HT have been shown that can mediate insect hemocytic immune reactions to infections and invasions. Here, we showed that TA increase hemocyte spreading in the rice stem borer, Chilo suppressalis. Furthermore, we cloned a cDNA encoding a tyramine receptor type 2 from the hemocytes in the C. suppressalis, viz., CsTA2, which shares high sequence similarity to members of the invertebrate tyramine receptor family. The CsTA2 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with TA induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in cells, with an EC50 value of 18.7 ± 5.3 nM, whereas OA, DA, 5-HT and other potential agonists did not have this response. The mRNA is present in various tissues including nerve cord, hemocytes, fat body, midgut, Malpighian tubules, and epidermis in the larval stage. Western blot analysis and immunohistochemistry assay displayed that CsTA2 was detected and presented on hemocytes. We also showed that TA induced Ca2+ release from the hemocytes of C. suppressalis. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Calcium (Ca2+) acts as a universal second messenger with a pivotal role in many cell types (Oh-hora and Rao, 2008). In cells of the immune system, Ca2+ signal is essential for diverse cellular functions, such as proliferation, differentiation, effector function, and a variety of transcriptional programs (Vig and Kinet, 2009; Wu et al., 2013a). The importance of intracellular free Ca2+ levels in mammalian signal transduction pathways is well documented (Oh-hora, 2009; Oh-hora and Rao, 2008). In recent years, researches about the Ca2+ signaling in the insect immunocytes (hemocytes) were frequently reported. Previous studies indicated that octopamine (OA) could induce the Ca2+ release and mediate cellular immune response in the insect hemocytes (Baines and Downer, 1994; Dunphy and Downer, 1994; Kim et al., 2009). Recent work also showed that OA can exert biphasic effects on ⇑ Corresponding authors at: State & Local Joint Engineering Research Center of Green Pesticide Invention and Application, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China (S.-F. Wu). E-mail addresses: [email protected] (S.-F. Wu), [email protected] (G.-Y. Ye). http://dx.doi.org/10.1016/j.jinsphys.2015.03.004 0022-1910/Ó 2015 Elsevier Ltd. All rights reserved.

cellular immune responses in insect hemocytes and these effects were mediated by CsOA1, an a-adrenergic-like OAR on the hemocytic membrane (Huang et al., 2012). This OAR appears to act through both Ca2+ and cAMP signaling pathways. However, we have no information regarding the involvement of tyramine (TA) in the immune response. OA and TA are structurally similar biogenic amines that are derived from the amino acid tyrosine (Roeder, 2005; Wu et al., 2010). OA and TA are trace amines in vertebrates. OA has been shown to regulate various physiological functions and behavioral process in insects, such as male aggression (Hoyer et al., 2008), locomotion and dance behavior (Barron et al., 2007; Fuchs et al., 2014; Wu et al., 2012), fight and flight (Roeder, 1999), egg laying (Fuchs et al., 2014; Wong and Lange, 2014), immune response (Adamo, 2014; Huang et al., 2012), and learning and memory (Mizunami et al., 2009). However, the physiological roles of TA, the metabolic precursor of OA, has been largely neglected. In recent years, many studies have supported the hypothesis that TA is not only a OA precursor but also a genuine neuroactive chemical in a variety of physiological processes in insects (Lange, 2009). It was reported that increases in TA levels could inhibit

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insect oviposition (Cossío-Bayúgar et al., 2012; Monastirioti et al., 1996). Co-injection of TA with DOPA, the precursor of melanin, had a strong cumulative negative effect on mosquito locomotion and survival (Fuchs et al., 2014). In addition of this, specific neurons that only contained TA, but not OA, were identified in Drosophila (Nagaya et al., 2002). All of these studies indicated that TA could as an independent neurotransmitter in insects. The effects of both OA and TA are mediated via the activation of G protein-coupled receptors (GPCRs), the stimulation of which activates a second-messenger cascade. Until now, many octopamine and tyramine receptors have been cloned from several insect species (Farooqui, 2012; Huang et al., 2007). The first potential insect TA receptor (TAR) was cloned from Drosophila melanogaster (Saudou et al., 1990). This receptor was firstly named as an OA receptor (OAR). However, TA was found to be 13–33-fold more potent than OA in the studies of both ligand binding and attenuation of adenylate cyclase activity (Arakawa et al., 1990; Robb et al., 1994; Saudou et al., 1990). Hence, this receptor was considered as tyramine receptor type 1 (TyR1). A member of the second class of TyR, CG7431, was also cloned from D. melanogaster (Cazzamali et al., 2005). When CG7431 was expressed in Chinese Hamster Ovary (CHO) cells or Xenopus oocytes, TA most potently increased intracellular calcium while other biogenic amines (up to 100 lM) or neuropeptides (up to 10 lM) have no effects (Cazzamali et al., 2005). An orthologous receptor was also characterized in Bombyx mori (Huang et al., 2009). A recent work showed a gene (CG16766) from D. melanogaster, represents a new group of tyramine receptors, which was designated as the Tyramine 3 receptors (TyR3) (Bayliss et al., 2013). This receptor has a different signal transduction and pharmacological profile to those of CG7431. Besides inducing [Ca2+]i response, incubation CG16766-expressing CHO cells with TA can also reduce forskolin-stimulated [cAMP]i (Bayliss et al., 2013). In the present study, the tyramine receptor type 2 (CsTA2) from Chilo suppressalis has been characterized. The gene was widely expressed in different developmental stages and various tissues, including hemocytes, fat body, midgut, epidermis and, more specifically, Malpighian tubules and nerve cord. Using HEK-293 cells stably transfected with CsTA2, we compared the cellular response to biogenic amines and selected synthetic agonists. Considering various roles of TA in physiological processes, CsTA2 expressed in the hemocytes might play an important role in insect immune response. Western blot analysis and immunohistochemistry assay confirmed that the protein of CsTA2 is expressed in C. suppressalis hemocytes. Finally, to verify the functional expression of the CsTA2, Ca2+ release by TA activation was assessed using C. suppressalis hemocytes in vitro.

hemolymph from twenty fifth instar larvae of rice stem borer was collected into Grace’s medium (1:10, v/v; Invitrogen, Carlsbad, CA). Hemocyte monolayer was prepared with 50 ll of hemocyte suspension containing 5 ll of test chemical (tyramine) or solvent (control) and this mixture was incubated in a 96 well tissue culture plate (Nunc, Roskilde, Denmark) for 20 min at 25 °C. The extent of hemocyte spreading was determined by counting the number of cells that displayed cytoplasmic expansion under a phase contrast microscope at 400 magnification. 2.3. Cloning of full-length CsTA2 cDNA Total RNA was isolated from the hemocytes of twenty fifth instar larvae of C. suppressalis using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The single-strand cDNA, synthesized from the RNA using a ReverTra Ace-a-kit (Toyobo, Osaka, Japan), was used as a template for PCRs. Degenerate primers (TA2-F and TA2-R, Table 1) corresponding to the highly conserved regions of tyramine receptors type 2 in B. mori (GenBank: BAI52937) and D. melanogaster (GenBank: NP_ 650652) were designed to amplify cDNA fragments. A fragment was isolated, ligated into the pGEM-T vector (Promega, Madison, WI) and subsequent sequencing confirmed that this is the expected CsTA2 partial sequence. To obtain the full length of CsTA2, 50 -RACE and 30 -RACE were performed using the 50 -Full RACE Kit and 30 -Full RACE Core Set Ver.2.0 (Takara, Shiga, Japan). RACE primers were designed according to the previous sequenced fragment (Table 1). Finally, the complete ORF of CsTA2 was obtained using a set of primers, TA2-compF and TA2-compR, and was confirmed to be the target gene by direct DNA sequencing. 2.4. Sequence analysis and phylogenic tree construction Nucleotide sequences were assembled and the open reading frame (ORF) was identified by DNAStar software package (Version 5.02). Sequence similarity/annotations and searching for

Table 1 The primers used in this study. Primers Degenerate primers TA2-F(598) TA2-R(840) For cDNA cloning 50 RACE outer primer 50 RACE inner primer 30 RACE outer primer 30 RACE inner primer 50 RACE-A 50 RACE-B 30 RACE-A 30 RACE-B TA2-compF TA2-compR

2. Materials and methods 2.1. Insect rearing and chemicals C. suppressalis were collected from field in Fuyang, China (119.6°E, 30.5°N), and were reared in laboratory for several generations with artificial diet (Han et al., 2012). The rearing conditions were 28 ± 1 °C, >80% relative humidity and a 16:8 h light dark photoperiod. (±)-Octopamine hydrochloride, tyramine hydrochloride, dopamine hydrochloride, serotonin hydrochloride, naphazoline hydrochloride, clonidine hydrochloride, forskolin, G418 disulfate salt, 3-isobutyl-1-methylxanthine (IBMX) and adenosine 50 -triphosphate salt (ATP) were all obtained from Sigma–Aldrich (St. Louis, MO, USA).

For real-time PCR TA2-RTF TA2-RTR Elongation factor-1 (EF-1)-F Elongation factor-1 (EF-1)-R

50 -CTCTGYACNGCNTCCATHCT-30 50 -AARAASGANCCCATNGCSGARAA-30 50 -CATGGCTACATGCTGACAGCCTA-30 50 -CGCGGATCCACAGCCTACTGATGATCAGTCGATG-30 50 -TACCGTCGTTCCACTAGTGATTT-30 50 -CGCGGATCCTCCACTAGTGATTTCACTATAGG-30 50 -CGGCACACCCCTCCTTGGTTGTGGTCT-30 50 -CCACACGAAGAAGATCATGGTGAGGGCA-30 50 -CCACCGATGTTGGGATGGTATGAGCC-30 50 -CAACCAAGGAGGGGTGTGCCGATAC-30 50 -ATGATCTCAATACCTGATAACGG-30 50 -TTATCGCTTATTCCTTTTACAT-30 50 -TCGCCAGGAGACACCAACAACTA-30 50 -GAAGCCTCCTACCACCAAACTCA-30 50 -TGAACCCCCATACAGCGAATCC-30 50 -TCTCCGTGCCAACCAGAAATAGG-30

For recombinant protein expression and eukaryotic expression TA2-BamHI-Fa 50 -GCGGATCCGCGAGGATATCTTGCGTAGT-30 TA2-EcoRI-Ra 50 -GCGAATTCTTACTTCATCGAATGCGTTCGGT-30

2.2. Hemocyte-spreading assay Hemocyte-spreading assay was modified from an established method (Huang et al., 2012; Srikanth et al., 2011). Briefly,

Primer sequence

a

TA2-HindIII-Fa

50 -CGGAAGCTTACCATGATCTCAATACC-30

TA2-XhoI-Ra

50 -GGCTCGAGTTATCGCTTATTCCTTT-30

Restriction sites are underlined.

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orthologous genes were performed using BLAST programs from the NCBI. Multiple sequence alignments of the complete amino acid sequences were performed with Clustal W2 (http://www.ebi.ac. uk/Tools/msa/clustalw2/). The transmembrane segments and topology of CsTA2 were predicted by TMHMM 2.0 (http://www. cbs.dtu.dk/services/TMHMM-2.0/). A phylogenetic tree was constructed with the neighbor-joining method, using 1000-fold bootstrap resampling, and the resulting tree was displayed graphically using MEGA 5.0 (Tamura et al., 2011). The Homo sapiens rhodopsin was used as an out-group. 2.5. Tissue distribution of CsTA2 For CsTA2 tissue expression, total RNAs were extracted from different developmental stages and tissues of C. suppressalis with Trizol reagent (Invitrogen) according to manufacturer’s instructions. Residual genomic DNA was removed by RQ1 RNase-Free DNase (Promega). Total RNA (1 lg) was reverse transcribed to cDNA with the ReverTra Ace-a-kit. For hemocytes collection, fifth instar naive larvae were surface sterilized with 70% ethanol and total hemolymph was collected by cutting its proleg. Nerve cords, fat body, midguts, Malpighian tubules and epidermis of the fifth instar larvae were dissected with the aid of stereomicroscope (Leica, Wetzlar, Germany) in cooled saline solution. Then all samples were immediately deep frozen in liquid nitrogen and preserved at 80 °C until analysis. 2.6. Antibody production and purification The anti-CsTA2 antiserum was raised against a fusion protein containing part of the third cytoplasmic loop (CPL3) of CsTA2 (Fig. 2). The cDNA fragment was amplified by PCR with specific primers (TA2-BamHI-F and TA2-EcoRI-R, Table 1). The fragment was cloned into pET28a vector (Merck, Germany) and over-expressed (HIS-CsTA2-CPL3). The polyclonal rabbit antiserum was raised commercially (HuaAn Biotechnology Co., Ltd., Hangzhou, China). An antibody to CsTA2 was purified from antiserum using the Montage Antibody Purification kit with PROSEP-A media (Millipore, Billerica, MA), following the manufacturer’s protocol. 2.7. Western blot analysis Hemolymph was collected from naïve larvae of C. suppressalis, mixed with two volumes of pre-cooled PBS buffer (pH 7.2), and centrifuged at 1000 g for 10 min at 4 °C. After discarding the plasma suspension, hemocyte precipitate were collected and washed with PBS buffer. Membrane proteins were isolated with a ProteoExtract Transmembrane Protein Extraction Kit (Novagen, Madison, WI) according to the manufacturer’s instructions. Total protein samples (10 lg each) were subjected to SDS–PAGE with 4% stacking gel and 12% separating gel, and proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (Sigma, St. Louis, MO) by a semi-dry electrophoretic transfer system (Bio-Rad). Membranes were blocked with 5% dry milk in Tris-buffered saline containing Tween 20 (TBS-T: 10 mM Tris– HCl, pH 7.5, 150 mM NaCl, 0.01% Tween 20) (TBST) overnight at 4 °C. Then membranes were washed with TBS-T three times and subsequently incubated with CsTA2 antibody (diluted 1:500 with 5% non-fat milk in TBS-T) for 1 h at room temperature. Membranes were washed with TBS-T, followed by incubation with a secondary antibody conjugated to horseradish peroxidase (Promega) (diluted 1:5000 with dry milk in TBS-T) for 1 h at room temperature. Signals were visualized with an enhanced chemiluminescence detection system (Super Signal West Pico Chemiluminescent Substrate; Pierce, Rockford, IL).

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2.8. Immunolocalization analysis To immune-localize CsTA2 in hemocytes, an aliquot of 30 ll hemolymph was collected from three C. suppressalis larvae and mixed with 170 ll Grace’s Insect medium (Invitrogen) containing 50 lg/ml tetracycline and 2 ll saturated 2-phenylthiourea (PTU). The diluted hemolymph was added to each well of a 8-well Lab-Tek chambered coverglass (Nunc, Thermo Fishers, Rochester, NY) and hemocytes were allowed to adhere to the coverglass for 20 min at 25 °C to form monolayers. Immuno-localization assay was performed as described previously (Wu et al., 2013a). Briefly, the monolayers were fixed in 4% paraformaldehyde (freshly prepared in PBS, pH 7.4) for 15 min at room temperature. After rinsing in PBS, hemocytes were blocked in 3% BSA for 1 h at room temperature. Then, the hemocytes were incubated with anti-CsTA2 antibody (diluted 1:200 in PBS containing 0.5% BSA) for 1 h at room temperate, or with pre-immune rabbit serum as the control. Subsequently, the hemocytes were thoroughly washed in PBS three times, and incubated with secondary goat anti-rabbit antibody conjugated with fluorescein isothiocyanate (FITC) (diluted 1:100 in PBS containing 0.5% BSA) for 45 min at room temperature. The hemocytes were stained with 40 -6-diamidino-2-phenylindole (DAPI, 1 lg/ml in PBS, Beyotime Biotech, Jiangsu, China) for 5 min. After thoroughly washed in PBS, the hemocytes were observed and photographed by fluorescent microscope (Zeiss, Göttingen, Germany). 2.9. Construction of expression plasmids An expression-ready construct of CsTA2 cDNA was generated by PCR, which was performed with specific primers (TA2-HindIII-F and TA2-XhoI-R, Table 1). The PCR product was digested with HindIII and XhoI. The digested DNA fragments were then subcloned into pcDNA3 vector (Invitrogen) yielding pcDNA3-CsTA2. The correct insertion was confirmed by DNA sequencing. 2.10. Cell culture, transfection, and creation of stable cell lines HEK-293 cells were grown in Dulbecco’s modified Eagle’s medium (D-MEM) (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL) at 37 °C and 5% CO2. PcDNA3-CsTA2 vector was introduced into the HEK 293 cells using Lipofectamine 2000 (Invitrogen). Stably transfected cells were selected in the presence of the antibiotic G418 at 0.8 mg mL1. After 2 weeks of G418 selection, G418-resistant colonies were trypsinized in cloning cylinders and transferred to 24-well plastic plates for expansion. These individual cell lines were analyzed for integration of the receptor DNA by RT-PCR. The clonal cell line most efficiently expressing CsTA2 was chosen for this study. 2.11. Ca2+ and cAMP assays [Ca2+]i was estimated and analyzed as previously described (Huang et al., 2012; Wu et al., 2013a). Briefly, HEK-293 cells stably expressed CsTA2 were seeded on the coverslip with D-MEM and incubated for 16–24 h at 37 °C and 5% CO2. Similarly, hemocytes from the fifth instar larvae were also seeded on the coverslip with Grace’s insect medium and incubated for 20 min at 25 °C to form monolayers. The cells were washed twice with Ringer’s buffer (155 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl26H2O, 2 mM NaH2PO46H2O, 10 mM glucose and 10 mM HEPES, pH 7.2). After washing, the cells were loaded with the fluorescent probe Fura 2-AM (Dojindo Laboratories, Kumamoto, Japan) using 0.2% Cremophor EL (Sigma–Aldrich) in D-MEM for 30 min at 37 °C. The coverslips were transferred to a microscopic chamber that was constantly perfused with Ringer’s buffer at 2 ml/min. The

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Fura-2 fluorescence at 510 nm by excitation at 340 or 380 nm with a xenon lamp was measured with individual cells using an Easy Ratio Pro calcium imaging system (PTI, Birmingham, NJ). The cAMP assay was performed as previously described (Wu et al., 2014, 2012). 2.12. Statistical analysis All studies were performed in four to six independent replicates and plotted by mean ± standard error using Origin software (Microcal, Northampton, MA). Statistical significance was determined by using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.

3. Results 3.1. Tyramine enhance hemocyte spreading Our results showed that TA enhanced hemocyte spreading with dose-dependent manner (Fig. 1). The stimulating effect of TA on hemocyte spreading began to show at 10 nM concentration. Although the dose-dependent increase of TA effect was not detected at the range of 10–100 nM, it showed obviously increase at the range of 100 nM to 1 lM. Cell spreading increased from about 20% in control and in experiment conducted in the presence of 1 nM TA concentration to about 45% in preparations exposed to 10 lM TA. 3.2. cDNA cloning and sequence analysis of CsTA2

Fig. 1. Effects of tyramine on hemocytes spreading. Each histogram bar represents means ± standard error (n = 5). SE bars annotated with the same letters are not significantly different (P > 0.05).

The entire coding sequence of potential tyramine receptor was amplified from C. suppressalis hemocytes using RACE and RT-PCR (Fig. S1. This sequences were designated CsTA2 (Fig. 2), according to its similarity to other insect tyramine receptors type 2 (Verlinden et al., 2010). The ORF (1422 bp) encodes a protein of amino acids with a calculated molecular weight of 53.6 kDa (GenBank accession number ADK91078), containing seven transmembrane domains (TMs). A TGA stop codon preceding the start codon was found at position 33 to 31 of the nucleotide sequence (Fig. S1). Three consensus sites for N-linked glycosylation are located in the N-terminal (N35, N57 and N62) (Fig. 2). In addition, several serine residues positioned in the intracellular loops were identified as sites for phosphorylation by cAMP-dependent Protein Kinase C (Fig. 2). The encoded protein of CsTA2, similar with BmTA2, had shorter extracellular N-terminus and shorter ICL 3 regions, compared with DmTA2 (Fig. 2). Amino acid sequence comparisons between CsTA2 and other insect biogenic amine receptors show high amino acid similarity with BmTA2, 86% (Huang et al., 2009), and DmTA2, 74% (Cazzamali et al., 2005). In order to determine the subfamily to which the CsTA2 is most similar, a phylogenetic family tree was created by comparing the amino acid

Fig. 2. Amino acid sequence alignment of CsTA2 and orthologous receptors from Bombyx mori (BmTA2; GenBank accession number BAI52937) and Drosophila melanogaster (DmTA2; accession number NP_650652). The amino acid position is indicated on the right. The predicted seven transmembrane regions are indicated by TM1–7. The alignment was performed by Clustal X and the shaded sequences highlight the identity level of amino acids between the receptors. Potential N-glycosylation sites and potential phosphorylation sites for protein kinase C are labeled by filled circles and asterisks, respectively. Conserved cysteine residues in the first and second extracellular loops are labeled with empty circles. The aspartic acid residue (D157) and the serine residues (S240and S244) that are predicted to be involved in agonist binding are labeled with filled triangles and filled quadrilaterals. The second phenylalanine after the FxxxWxP motif in TM6, which is a unique feature of aminergic receptors is indicated by a rhombus. Underlined letters represent CPL3 from which the CsTA2-specific antigen was derived.

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Fig. 3. Phylogenetic tree of CsTA2 and various biogenic amine receptors. Maximum likelihood trees were constructed using MEGA5 software with 1000-fold bootstrap resampling. The numbers at the nodes of the branches represent the level of bootstrap support for each branch. Human rhodopsin receptor was used as the outgroup. The receptor sequences followed by their GenBank accession numbers are listed in the order illustrated: C. suppressalis (CsTA2, No. ADK91078, this work)); B. mori (BmTA2); D. melanogaster (DmTA2); A. mellifera (AmTA2, No. NP_001032395); C. suppressalis (CsOA1, No. AEQ33589); B. mori (BmOA1, No. NP_001091748); P. americana (PeaOA1, No. AAP93817); A. mellifera (AmOA1, No. NP_001011565); D. melanogaster (DmOA1, No. NP_732541); A. mellifera (AmTA1, No. NP_001011594); D. melanogaster (DmTA1, No. BAB71788); C. suppressalis (CsTA1, No. JQ416145); B. mori (BmTA1, No. BAD11157); P. americana (PaTA1, No. AM990461); C. suppressalis (CsOA2B2, No. JN620367); B. mori (BmOAR2, No. BAJ06526); D. melanogaster (DmOA2B2, No. AJ880689); A. mellifera (AmOA2B2, No. XP_396348); A. mellifera (AmOA2B3, No. XP_003249152); D. melanogaster (DmOA2B3, No. AJ884591); C. suppressalis (CsOA2B3, No. KF640248); D. melanogaster (DmOA2B1, No. AJ880687); C. suppressalis (CsOA2B1, No. KF640249); A. mellifera (AmOA2B1, No. XP_397139); A. mellifera (AmOA3, No. XP_001122075); D. melanogaster (DmOA3, No. NP_650754); C. suppressalis (CsOA3-L, No. KF460457); C. suppressalis (CsOA3-S, No. KF460458); H. sapiens rhodopsin (human rhodopsin receptor, No. NP_000530).

sequence of CsTA2 with those of known biogenic amine receptors from different insect species. CsTA2 clustered with BmTA2, DmTA2 and AmTA2 in a distinct clade, which belongs to a new family of tyramine receptor (Cazzamali et al., 2005) (Fig. 3). 3.3. Expression pattern of CsTA2 CsTA2 was expressed in all developmental stages including egg, larva, pupa and adult (Fig. 4A). At the fifth instar larval stage, CsTA2 was expressed in all tested tissues but was highly expressed in the nerve cord and Malpighian tubules, followed by the hemocytes, fat body, epidermis and midgut including hemocytes, fat body, midgut, nerve cord and epidermis (Fig. 4B). 3.4. Functional characterization of CsTA2 A HEK-293 cell line stably expressing CsTA2 was generated in order to examine the functional properties of the receptor. The tested compounds included biogenic amines (TA, OA, DA, 5-HT)

and OAR agonists (clonidine, naphazoline). TA produced Ca2+ responses at concentrations ranging from 1 nM (occasionally observed) to 300 nM (maximal response), and its EC50 value was estimated at 18.7 ± 5.3 nM (Fig. 5A). Apart from TA, no compounds showed Ca2+ elevations at 10 lM concentrations (Fig. 5B). Therefore, CsTA2 was selectively activated by TA and coupled with intracellular calcium mobilization. We determined [cAMP]i in order to investigate whether CsTA2 was coupled to other G proteins. The results showed no significant changes in [cAMP]i following application of 100 lM TA alone or together with 10 lM forskolin, compared with each control (Fig. 6). This results suggested that CsTA2 is not coupled to the Gs or Gi protein, which stimulates or inhibits adenylate cyclase, respectively. 3.5. Localization and functional expression of CsTA2 in C. suppressalis hemocytes To detect CsTA2 in hemocytes, membrane proteins from naïve hemocytes were prepared and analyzed by SDS–PAGE, and CsTA2

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was identified by immunoblotting using rabbit polyclonal antibody to CsTA2. A protein band around 53.5 kDa was detected in the membrane extract of hemocytes (Fig. 7A), suggesting that CsTA2 was indeed expressed in hemocytes. To determine the location of CsTA2 in hemocytes, an immune-localization assay was conducted. Hemocytes were collected from naïve larvae, fixed by 4% paraformaldehyde and blocked with BSA. These fixed hemocytes were then incubated with rabbit polyclonal antibody against the CsTA2 (Fig. 7B, panel b). The results showed that green fluorescence signal was detected in the membrane of most hemocytes including granulocytes (GR) and plasmatocytes (PL) (Fig. 7B, panel d). To examine the functional expression of CsTA2 in the hemocyte, we conducted the fluorescence imaging of [Ca2+]i in vitro. The results showed that 100 nM TA could induce calcium release from rice stem borer hemocytes (Fig. 8). 4. Discussion

Fig. 4. Expression pattern of CsTA2 analyzed by RT-PCR and qPCR. (A) Expression pattern of CsTA2 in different developmental stages. (B) Expression pattern of CsTA2 mRNA levels in tissues of fifth instar larvae were quantified by qPCR and normalized against levels of Elongation factor-1 (EF-1) mRNA. Tissues tested are hemocytes (HC), fat body (FB), midgut (MG), Malpighian tubules (MT), epidermis (EP) and nerve cord (NC). Data represent means ± standard error (n = 3 repetitions).

Fig. 5. Representative Ca2+ responses for TA and various agonists in CsTA2/HEK-293 cells. (A) Dose–response curve for TA, as obtained from Ca2+ assays. The data are shown as a percentage of the response to 100 nM TA. (B) Effects of natural biogenic amines and potential agonists on [Ca2+]i in CsTA2/HEK-293 cells.

Until now, four aminergic receptors have been cloned and experimentally examined from C. suppressalis: three OARs: CsOA1 (Huang et al., 2012), CsOA2B2 (Wu et al., 2012) and a novel family of OAR (CsOA3) (Wu et al., 2014); and only one tyramine receptor, CsTA1 (Wu et al., 2013b). In the present study, we describe the molecular characteristics, expression pattern and functional properties of another tyramine receptor from the rice stem borer, C. suppressalis. Orthologous receptors have been previously isolated from D. melanogaster (Cazzamali et al., 2005) and B. mori (Huang et al., 2009). The phylogenetic result indicates that insect TA2 is not closely related to other insect octopamine or tyramine receptors (Fig. 3). Our results also indicate that CsTA2 was specifically activated by tyramine (TA) and coupled to intracellular calcium mobilization. The distribution pattern of CsTA2 revealed a ubiquitous expression in all developmental stages and examined tissues (Fig. 4). This pattern of CsTA2 expression is in agreement with TA regulating a plethora of physiological functions not only in the nerve system (Roeder, 2005) but also in the peripheral tissues, such as antenna (Brigaud et al., 2009; Duportets et al., 2010), salivary gland (Rotte et al., 2009) and Malpighian tubule (Herman and Blumenthal, 2006). However, qPCR results indicated that CsTA2 is expressed predominantly in the nervous cord of the fifth instar larvae, suggesting a putative role in the modulation of neuronal activity in accordance with the action of OA and TA as neurotransmitter described in insects. Our results showed that CsTA2 was highly

Fig. 6. Effects of TA on basal and forskolin-stimulated [cAMP]i in CsTA2/HEK-293 cells. Cells were treated with 100 lM TA in the presence or absence of 10 lM forskolin for 20 min at 37 °C, and [cAMP]i was determined. Data represent the means ± standard error of three independent experiments, performed in duplicate. FK, forskolin.

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D. melanogaster (Blumenthal, 2009), which might interact with TyR2 to perform this physiological functions. CsTA2 was stably expressed in HEK-293 cells, which have been used successfully in previous studies to examine the functional properties of cloned insect TARs (Balfanz et al., 2014; Huang et al., 2007, 2008, 2010, 2009; Wu et al., 2012). CsTA2 showed higher specificity for TA than for other biogenic amines in both binding and Ca2+ mobilization assays. The potency of TA on CsTA2 was similar to that in BmTA2 (11.6 nM in HEK-293 cells) (Huang et al., 2009) and higher than DmTA2 (500 nM in CHO cells and 300 nM in Xenopus oocytes) (Cazzamali et al., 2005) in terms of EC50 values. In agonist assays, all of other biogenic amines (OA, DOP and 5-HT) and four full octopamine receptor agonists (amitraz, chlordimeform, clonidine and naphazoline) exhibited no agonist activity. However, 100 lM OA and DOP were capable of inducing Ca2+ responses via BmTA2 (Huang et al., 2009). The differences between CsTA2 and BmTA2 could be due to differences in receptors, the expression systems, and/or expression levels on the cell membrane. The innate immune system of insects is divided into humoral and cellular defense responses (Strand, 2008). Cellular defenses refer to hemocyte-mediated responses such as phagocytosis and encapsulation. Hence, hemocytes have been shown to be important in insect innate immunity (Lavine and Strand, 2002). OA and 5-HT have been known to enhance hemocytic phagocytosis and nodule formation during bacterial invasion (Dunphy and Downer, 1994; Kim et al., 2009). Especially, OA could increase secondary messengers, such as intracellular free Ca2+ (Huang et al., 2012; Jahagirdar et al., 1987) and inositol triphosphate in hemocytes (Baines and Downer, 1994). The role of Ca2+ in immunity of vertebrate, including controls of proliferation, differentiation and transcription events in different immune cells have been extensively studied by various authors (Oh-hora and Rao, 2008; Vig and Kinet, 2009). In insects, it was showed that cytokines, such as growth-blocking peptide (or plasmatocytes-spreading peptide), which mediate insect immunity also depends on intracellular Ca2+ (Srikanth et al., 2011; Tsuzuki et al., 2014). Our recent works indicated that octopamine receptor type 1 (CsOA1) (Huang et al., 2012) and ryanodine receptor (Wu et al., 2013a) were presented in insect hemocytes. All of these receptors could couple with intracellular Ca2+. In this study, we showed that TA could stimulate hemocyte spreading, an initiating event in cellular immunity and detected that the mRNA and protein of CsTA2 in the hemocyte of

Fig. 7. Immunoblot analysis of CsTA2 in the membrane extract of hemocytes and localization of CsTA2 on hemocytes. (A) Immunoblot analysis of CsTA2 in the membrane extract of hemocytes. Membrane extracts of hemocytes were prepared as described. The arrow indicates CsTA2 (53.5 kDa) in the membrane extract of hemocytes. (B) Immuno-localization of CsTA2 on hemocytes. Hemolymph was collected from naïve larvae and mixed with Grace’s medium. Diluted hemolymph was added to a 12-well multi-test slide and hemocytes were allowed to adhere for 15–20 min. Hemocytes were fixed with paraformaldehyde and blocked with BSA. Then the fixed hemocytes were incubated with rabbit polyclonal antibody to the CsTA2. CsTA2 on membrane of hemocytes was visualized by FITC-labeled secondary antibody (green) (b), and the nuclei of hemocytes were stained with DAPI (blue) (c). Differential interference contrast (DIC) view of hemocytes (a); merged image of DAPI and FITC (b and c). Scale bar = 10 lm. Granulocytes (GR) and plasmatocytes (PL) was showed by white arrows. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

expressed in the Malpighian tubules. Together with this, DmTA2 was also abundantly expressed in the Malpighian tubule of D. melanogaster (Herman and Blumenthal, 2006). Moreover, TA is a potent diuretic factor when applied to the Malpighian tubule of

Fig. 8. Representative Ca2+ responses for 100 nM TA on Chilo suppressalis hemocytes.

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C. suppressalis. The future work is to study the mechanism of TA and CsTA2 expressed in hemocytes how to be involved in cellular immune responses during infection. Acknowledgements This work was financed by China National Science Fund for Innovative Research Groups of Biological Control (Grant No. 31021003), National Natural Science Foundation of China (Grant No. 31401782) and China National Science Fund for Distinguished Young Scholars (Grant No. 31025021). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jinsphys.2015.03. 004. References Adamo, S.A., 2014. The effects of stress hormones on immune function may be vital for the adaptive reconfiguration of the immune system during fight-or-flight behavior. Integr. Comp. Biol. 54, 419–426. Arakawa, S., Gocayne, J.D., McCombie, W.R., Urquhart, D.A., Hall, L.M., Fraser, C.M., Venter, J.C., 1990. Cloning, localization, and permanent expression of a Drosophila octopamine receptor. Neuron 4, 343–354. Baines, D., Downer, R.G.H., 1994. Octopamine enhances phagocytosis in cockroach hemocytes: involvement of inositol trisphosphate. Arch. Insect Biochem. Physiol. 26, 249–261. Balfanz, S., Jordan, N., Langenstück, T., Breuer, J., Bergmeier, V., Baumann, A., 2014. Molecular, pharmacological, and signaling properties of octopamine receptors from honeybee (Apis mellifera) brain. J. Neurochem. 129, 284–296. Barron, A.B., Maleszka, R., Vander Meer, R.K., Robinson, G.E., 2007. Octopamine modulates honey bee dance behavior. Proc. Natl. Acad. Sci. U.S.A. 104, 1703–1707. Bayliss, A., Roselli, G., Evans, P.D., 2013. A comparison of the signalling properties of two tyramine receptors from Drosophila. J. Neurochem. 125, 37–48. Blumenthal, E.M., 2009. Isoform- and cell-specific function of tyrosine decarboxylase in the Drosophila malpighian tubule. J. Exp. Biol. 212, 3802–3809. Brigaud, I., Grosmaître, X., François, M.-C., Jacquin-Joly, E., 2009. Cloning and expression pattern of a putative octopamine/tyramine receptor in antennae of the noctuid moth Mamestra brassicae. Cell Tissue Res. 335, 455–463. Cazzamali, G., Klaerke, D.A., Grimmelikhuijzen, C.J.P., 2005. A new family of insect tyramine receptors. Biochem. Biophys. Res. Commun. 338, 1189–1196. Cossío-Bayúgar, R., Miranda-Miranda, E., Narváez Padilla, V., Olvera-Valencia, F., Reynaud, E., 2012. Perturbation of tyraminergic/octopaminergic function inhibits oviposition in the cattle tick Rhipicephalus (Boophilus) microplus. J. Insect Physiol. 58, 628–633. Dunphy, G.B., Downer, R.G.H., 1994. Octopamine, a modulator of the haemocytic nodulation response of non-immune Galleria mellonella larvae. J. Insect Physiol. 40, 267–272. Duportets, L., Barrozo, R.B., Bozzolan, F., Gaertner, C., Anton, S., Gadenne, C., Debernard, S., 2010. Cloning of an octopamine/tyramine receptor and plasticity of its expression as a function of adult sexual maturation in the male moth Agrotis ipsilon. Insect Mol. Biol. 19, 489–499. Farooqui, T., 2012. Review of octopamine in insect nervous systems. Open Access Insect Physiol. 4, 1–17. Fuchs, S., Rende, E., Crisanti, A., Nolan, T., 2014. Disruption of aminergic signalling reveals novel compounds with distinct inhibitory effects on mosquito reproduction, locomotor function and survival. Sci. Rep. 4. Han, L., Li, S., Liu, P., Peng, Y., Hou, M., 2012. New artificial diet for continuous rearing of Chilo suppressalis (Lepidoptera: Crambidae). Ann. Entomol. Soc. Am. 105, 253–258. Herman, A.M., Blumenthal, E.M., 2006. Identification of the tyramine receptor in the Drosophila Malpighian tubule. FASEB J. 20, 345–346. Hoyer, S.C., Eckart, A., Herrel, A., Zars, T., Fischer, S.A., Hardie, S.L., Heisenberg, M., 2008. Octopamine in male aggression of Drosophila. Curr. Biol. 18, 159–167. Huang, J., Hamasaki, T., Ozoe, F., Ohta, H., Enomoto, K.-I., Kataoka, H., Sawa, Y., Hirota, A., Ozoe, Y., 2007. Identification of critical structural determinants responsible for octopamine binding to the a-adrenergic-like Bombyx mori octopamine receptor. Biochemistry 46, 5896–5903. Huang, J., Hamasaki, T., Ozoe, F., Ozoe, Y., 2008. Single amino acid of an octopamine receptor as a molecular switch for distinct G protein couplings. Biochem. Biophys. Res. Commun. 371, 610–614.

Huang, J., Ohta, H., Inoue, N., Takao, H., Kita, T., Ozoe, F., Ozoe, Y., 2009. Molecular cloning and pharmacological characterization of a Bombyx mori tyramine receptor selectively coupled to intracellular calcium mobilization. Insect Biochem. Mol. Biol. 39, 842–849. Huang, J., Hamasaki, T., Ozoe, Y., 2010. Pharmacological characterization of a Bombyx mori a-adrenergic-like octopamine receptor stably expressed in a mammalian cell line. Arch. Insect Biochem. Physiol. 73, 74–86. Huang, J., Wu, S.F., Li, X.H., Adamo, S.A., Ye, G.Y., 2012. The characterization of a concentration-sensitive a-adrenergic-like octopamine receptor found on insect immune cells and its possible role in mediating stress hormone effects on immune function. Brain Behav. Immun. 26, 942–950. Jahagirdar, A.P., Milton, G., Viswanatha, T., Downer, R.G.H., 1987. Calcium involvement in mediating the action of octopamine and hypertrehalosemic peptides on insect haemocytes. FEBS Lett. 219, 83–87. Kim, G.S., Nalini, M., Kim, Y., Lee, D.W., 2009. Octopamine and 5-hydroxytryptamine mediate hemocytic phagocytosis and nodule formation via eicosanoids in the beet armyworm. Spodoptera exigua. Arch. Insect Biochem. Physiol. 70, 162–176. Lange, A.B., 2009. Tyramine: from octopamine precursor to neuroactive chemical in insects. Gen. Comp. Endocrinol. 162, 18–26. Lavine, M.D., Strand, M.R., 2002. Insect hemocytes and their role in immunity. Insect Biochem. Mol. Biol. 32, 1295–1309. Mizunami, M., Unoki, S., Mori, Y., Hirashima, D., Hatano, A., Matsumoto, Y., 2009. Roles of octopaminergic and dopaminergic neurons in appetitive and aversive memory recall in an insect. BMC Biol. 7, 46. Monastirioti, M., Linn, J., Charles, E., White, K., 1996. Characterization of Drosophila tyramine b-hydroxylase gene and isolation of mutant flies lacking octopamine. J. Neurosci. 16, 3900–3911. Nagaya, Y., Kutsukake, M., Chigusa, S.I., Komatsu, A., 2002. A trace amine, tyramine, functions as a neuromodulator in Drosophila melanogaster. Neurosci. Lett. 329, 324–328. Oh-hora, M., 2009. Calcium signaling in the development and function of T-lineage cells. Immunol. Rev. 231, 210–224. Oh-hora, M., Rao, A., 2008. Calcium signaling in lymphocytes. Curr. Opin. Immunol. 20, 250–258. Robb, S., Cheek, T., Hannan, F., Hall, L., Midgley, J., Evans, P., 1994. Agonist-specific coupling of a cloned Drosophila octopamine/tyramine receptor to multiple second messenger systems. EMBO J. 13, 1325. Roeder, T., 1999. Octopamine in invertebrates. Prog. Neurobiol. 59, 533–561. Roeder, T., 2005. Tyramine and octopamine: ruling behavior and metabolism. Annu. Rev. Entomol. 50, 447–477. Rotte, C., Krach, C., Balfanz, S., Baumann, A., Walz, B., Blenau, W., 2009. Molecular characterization and localization of the first tyramine receptor of the American cockroach (Periplaneta americana). Neuroscience 162, 1120–1133. Saudou, F., Amlaiky, N., Plassat, J.L., Borrelli, E., Hen, R., 1990. Cloning and characterization of a Drosophila tyramine receptor. EMBO J. 9, 3611–3617. Srikanth, K., Park, J., Stanley, D.W., Kim, Y., 2011. Plasmatocyte-spreading peptide influences hemocyte behavior via eicosanoids. Arch. Insect Biochem. Physiol. 78, 145–160. Strand, M.R., 2008. The insect cellular immune response. Insect Sci. 15, 1–14. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Tsuzuki, S., Matsumoto, H., Furihata, S., Ryuda, M., Tanaka, H., Jae Sung, E., Bird, G.S., Zhou, Y., Shears, S.B., Hayakawa, Y., 2014. Switching between humoral and cellular immune responses in Drosophila is guided by the cytokine GBP. Nat. Commun. 5. Verlinden, H., Vleugels, R., Marchal, E., Badisco, L., Pflüger, H.J., Blenau, W., Broeck, J.V., 2010. The role of octopamine in locusts and other arthropods. J. Insect Physiol. 56, 854–867. Vig, M., Kinet, J.-P., 2009. Calcium signaling in immune cells. Nat. Immunol. 10, 21–27. Wong, R., Lange, A.B., 2014. Octopamine modulates a central pattern generator associated with egg-laying in the locust. Locusta migratoria. J. Insect Physiol. 63, 1–8. Wu, S.F., Guo, J.Y., Huang, J., Ye, G.Y., 2010. Advances in insect octopamine and tyramine. Acta Entomol. Sin. 53, 1157–1166. Wu, S.F., Yao, Y., Huang, J., Ye, G.Y., 2012. Characterization of a b-adrenergic-like octopamine receptor from the rice stem borer (Chilo suppressalis). J. Exp. Biol. 215, 2646–2652. Wu, S.F., Wang, F., Huang, J., Fang, Q., Shen, Z.C., Ye, G.Y., 2013a. Molecular and cellular analyses of a ryanodine receptor from hemocytes of Pieris rapae. Dev. Comp. Immunol. 41, 1–10. Wu, S.F., Huang, J., Ye, G.Y., 2013b. Molecular cloning and pharmacological characterisation of a tyramine receptor from the rice stem borer, Chilo suppressalis (Walker). Pest Manag. Sci. 69, 126–134. Wu, S.F., Xu, G., Qi, Y.X., Xia, R.Y., Huang, J., Ye, G.Y., 2014. Two splicing variants of a novel family of octopamine receptors with different signaling properties. J. Neurochem. 129, 37–47.