Molecular characterization and expression profiles of nicotinic ...

Insect Science (2016) 0, 1–14, DOI 10.1111/1744-7917.12324

ORIGINAL ARTICLE

Molecular characterization and expression profiles of nicotinic acetylcholine receptors in the rice striped stem borer, Chilo suppressalis (Lepidoptera: Crambidae) Gang Xu1 , Shun-Fan Wu1,2 , Zi-Wen Teng1 , Hong-Wei Yao1 , Qi Fang1 , Jia Huang1 and Gong-Yin Ye1 1 State

Key Laboratory of Rice Biology & Key Laboratory of Agricultural Entomology of Ministry of Agriculture, Institute of Insect Sciences,

Zhejiang University, Hangzhou, China and 2 State & Local Joint Engineering Research Center of Green Pesticide Invention and Application, College of Plant Protection, Nanjing Agricultural University, Nanjing, China

Abstract Nicotinic acetylcholine receptors (nAChRs) are members of the cys-loop ligandgated ion channel (cysLGIC) superfamily, mediating fast synaptic cholinergic transmission in the central nervous system in insects. Insect nAChRs are the molecular targets of economically important insecticides, such as neonicotinoids and spinosad. Identification and characterization of the nAChR gene family in the rice striped stem borer, Chilo suppressalis, could provide beneficial information about this important receptor gene family and contribute to the investigation of the molecular modes of insecticide action and resistance for current and future chemical control strategies. We searched our C. suppressalis transcriptome database using Bombyx mori nAChR sequences in local BLAST searches and obtained the putative nAChR subunit complementary DNAs (cDNAs) via reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. Similar to B. mori, C. suppressalis possesses 12 nAChR subunits, including nine α-type and three β-type subunits. Quantitative RT-PCR analysis revealed the expression profiles of the nAChR subunits in various tissues, including the brain, subesophageal ganglion, thoracic ganglion, abdominal ganglion, hemocytes, fat body, foregut, midgut, hindgut and Malpighian tubules. Developmental expression analyses showed clear differential expression of nAChR subunits throughout the C. suppressalis life cycle. The identification of nAChR subunits in this study will provide a foundation for investigating the diverse roles played by nAChRs in C. suppressalis and for exploring specific target sites for chemicals that control agricultural pests while sparing beneficial species. Key words Chilo suppressalis; expression profiles; nAChRs

Introduction Acetylcholine (ACh) is considered to be the major excitatory neurotransmitter in the insect central nervous system, as demonstrated in flies, honeybees, locusts and Correspondence: Gong-Yin Ye, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China. Tel: +86 571 8898 2696; email: [email protected]

grasshoppers (Dupuis et al., 2012). ACh is also the most abundant neurotransmitter in the insect brain, especially in the olfactory system and sensory pathways (Dupuis et al., 2012). ACh released from the presynaptic terminal can activate two distinct types of receptors located in postsynaptic cells. These receptors are muscarinic acetylcholine receptors (mAChRs), belonging to the G-protein coupled receptors, and nicotinic acetylcholine receptors (nAChRs), belonging to the ligand-gated ion channels 1

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Authors Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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(Collin et al., 2013). The nAChRs are the members of a cys-loop ligand-gated ion channel (cysLGIC) superfamily mediating fast cholinergic synaptic transmission in the insect central nervous system (Lee & O’Dowd, 1999). They include five homologous subunits arranged around a central ion channel, which are encoded by several α-type and β-type subunit genes (Shao et al., 2007). Each subunit possesses an N-terminal extracellular region containing agonist binding sites, and the typical cys-loop motif consists of two disulphide bond-forming cysteines separated by 13 residues. This cys-loop motif is associated with the assembly of acetylcholine receptors and ion channel gating (Green & Wanamaker, 1997; Albuquerque et al., 2009). The ACh-binding sites are located at the interface of two vicinal subunits and are formed by six distinctive domains (loops A–F) in the N-terminal extracellular region (Jones & Sattelle, 2010). Each subunit possesses four transmembrane domains (TM1–TM4). The subunits contain two adjacent cysteine residues in loop C that are associated with ACh binding and are defined as α-type subunits, whereas the remaining subunits without the two cysteines are referred to as β-type subunits (Kao & Karlin, 1986). A large cytoplasmic loop between TM3 and TM4 possesses several phosphorylation sites that are associated with regulating receptor activity (Hopfield et al., 1988). Since the genome of the fruit fly Drosophila melanogaster was published in 2000 (Adams et al., 2000), the genome sequences of other insects have been continuously completed. This information has been used to characterize the nAChR subunit gene families of Anopheles gambiae (malaria mosquito) (Holt et al., 2002; Jones et al., 2005), Apis mellifera (honey bee) (Jones et al., 2006; Tribolium Genome Sequencing Consortium, 2006), Tribolium castaneum (red flour beetle) (Jones & Sattelle, 2007; Tribolium Genome Sequencing Consortium, 2008), Nasonia vitripennis (parasitoid jewel wasp) (Jones et al., 2010; Werren et al., 2010), Acyrthosiphon pisum (pea aphid) (Tribolium Genome Sequencing Consortium, 2010; Dale et al., 2010), and Bombyx mori (silk worm) (Xia et al., 2004; Shao et al., 2007). These seven insects represent a variety of orders that evolved approximately 300 million years ago, and the nAChR subunit gene families of these species are still compact (Jones & Sattelle, 2010). Due to the publication of several insect genomes, insect nAChRs have been identified, which will contribute to understanding the variety of roles played by nAChRs. However, only a limited amount of data is available regarding the involvement of nAChRs in insect behaviors and cognitive processes (Gauthier, 2010). The roles of nAChRs in honey bee behaviors have been reported in previous C 2016

studies. For instance, injection of nicotine, which is an nAChR agonist, revealed that the action potential of the cholinergic signaling system enhances short-term memory and acquisition (Thany et al., 2005). In contrast, injection of mecamylamine, which is an nAChR antagonist, suppresses memory recall or olfactory learning (Lozano et al., 2001). In addition, a distinctive nAChR subtype that is regarded as α-BGT (bungarotoxin) sensitive was shown to be associated with long-term memory, whereas another α-BGT insensitive subtype affected by mecamylamine is involved in retrieval processes (Dacher et al., 2005). In D. melanogaster, nAChR α7 plays an important role in escape behaviors due to its abundance in the dendrites of the huge fiber system (Fayyazuddin et al., 2006). Although little is known about the functional roles of nAChRs, studies exploiting nAChRs as insecticide targets have been well documented. In insects, nAChRs play an essential role in the central nervous system (Tomizawa & Casida, 2001). The exploitation of economically important insecticides targeting nAChRs is attributed to their great abundance within the central nervous system of insects. Neonicotinoid insecticides are selective agonists of insect nAChRs and have been widely applied in the field to control various insect pests (Jeschke & Nauen, 2008; Liu et al., 2008; Zhuang et al., 2015). Since the first neonicotinoid insecticide, imidacloprid, was introduced as a commercial insecticide in the early 1990s, it has shown the most rapid growth in sales among insecticides worldwide (Matsuda et al., 2001). Six other neonicotinoid chemicals also have been developed into insecticides, such as nitenpyram, acetamiprid, thiamethoxam, thiacloprid, clothianidin and dinotefuran (Shao et al., 2011). Moreover, a novel type of nAChR-targeting insecticide, the sites of which are different from imidacloprid, has been used to improve the efficiency of insect pest control. These insecticides are the spinosyns, which are derived from the fermentation products of the actinomycete bacterium Saccharopolyspora spinosa and were developed as commercial insecticides in 1997 (Sparks et al., 2001; Markussen & Kristensen, 2012). Spinosad is a natural mixture composed of two active compounds from S. spinosa: spinosyn A and spinosyn D. After exposure to spinosad, insects exhibit tremors and paralysis caused by neuromuscular fatigue as the insecticide disturbs the central nervous system, ultimately resulting in death (Salgado et al., 1997; Salgado, 1998). The rice striped stem borer (SSB), Chilo suppressalis (Walker) (Lepidoptera: Crambidae), is one of the most economically important rice pests in Asia, southern Europe and northern Africa (He et al., 2008). The rice stem borer larvae feed within plant stems and cause significant crop loss annually, particularly in China due to rice cultivation and the popularization of hybrid varieties (Wang

The Authors Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences, 0, 1–14

nAChRs in C. suppressalis

et al., 2016). Chemical control is still the major approach used to protect rice from damage by the rice stem borer. Unfortunately, C. suppressalis has developed high resistance to organophosphate and nereistoxin insecticides due to their sustained application in the field, and the estimated cost of controlling this pest is approximately US$160 million annually (Wu et al., 2013a; Chang et al., 2014; Su et al., 2014). However, insecticides are not always effective due to the narrow window for damage control between hatching and penetration into the plant stem of rice stem larvae (Wang et al., 2014). Although lepidopteran insects are very important in agriculture, the members of the nAChR subunit gene family in this order are still unclear to a large extent, except in the silkworm, B. mori and the codling moth, Cydia pomonella (Shao et al., 2007; Martin & Garczynski, 2016). In this study, we present both the molecular characterization and expression profiles of nAChR subunits from C. suppressalis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses determined that there are clear differential expression patterns of nAChR subunits in various tissues and throughout the C. suppressalis life cycle. To our knowledge, it is the most comprehensive report of nAChR subunits of a rice pest to date. Identification of nAChR subunits is a key step in investigating the diverse roles played by nAChRs in the C. suppressalis central nervous system and in identifying specific target sites for chemicals that control agricultural pests while sparing beneficial species. Materials and methods Insect rearing One C. suppressalis colony has been continuously reared in our laboratory from larvae that were originally collected from a rice field in Fuyang, Zhejiang Province, China, in 2012. The larvae are reared on an artificial diet (Han et al., 2012) and maintained at 25 ± 1°C, with approximately 80% relative humidity, under a 14 : 10 light : dark cycle. Identification of nAChR subunit genes C. suppressalis nAChR subunits were identified by screening larval central nervous system transcriptome data for the rice striped stem borer, which are available in the Sequence Read Archive (SRA) database, under accession number SRX1022691) (Xu et al., 2015). Basic Local Alignment Search Tool (BLAST) queries were performed using the amino acid sequences of nAChR subunits from the silkworm, B. mori. The BLAST + 2.2.23

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software (downloadable from the National Center for Biotechnology Information [NCBI], Bethesda, MD, USA; ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/) was used to conduct local BLAST queries to search the assembled unigenes. In C. suppressalis, 12 putative nAChR subunit sequences were identified in our transcriptome data. Each sequence putatively encoding an nAChR subunit was further identified through BLASTX searches against the non-redundant database at the NCBI (http://www.ncbi.nlm.nih.gov/). Cloning of full-length cDNAs of nAChR subunits To clone the full-length complementary DNAs (cDNAs) of 12 putative nAChR subunits, eight subunits with complete open reading frames (ORFs) and others without complete ORFs were confirmed and completed via RT-PCR and rapid-amplification of cDNA ends (RACE), respectively, with corresponding primers (Table S1). For the cloning of nAChR subunit cDNAs, the brains or fat bodies of fifth-instar larvae were dissected for RNA extraction in a phosphate-buffered saline solution containing a ribonuclease (RNase) inhibitor (TaKaRa, Kusatsu, Japan) and immediately frozen in liquid nitrogen, then stored at -80°C. Total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized from 1 μg of total RNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, Beijing, China). The isolated total RNA from the brain or fat body was used to synthesize the 3 - and 5 -RACE cDNA templates with the SMARTTM RACE cDNA Amplification kit (Clontech, Mountain View, CA, USA) (Zhu et al., 2001). Based on the partial sequence obtained from our C. suppressalis transcriptome data, gene-specific primers for RACE (Table S1) were designed starting 100 bp from the ends, to avoid low-quality sequences and possible vector sequence contamination. The 3 and 5 end sequences were further screened for validation through BLAST searches against the NCBI database. The full length of the nAChR subunits was amplified via RT-PCR using KOD-FX (Toyobo, Osaka, Japan). PCR amplification was conducted in a 50 μL reaction mixture containing 25 μL of 2× KOD FX buffer, 8 μL of deoxynucleotide triphosphate (dNTP) mix, 1 μL of KOD FX, 1 μL of the cDNA template and 2 μL of each primer. Sequence analysis of the nAChR subunits Multiple alignment of the derived amino acid sequences of the nAChR subunits was performed using

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the ClustalX2 program (Larkin et al., 2007) with default parameters. The phylogenetic tree was constructed with MEGA5.0 (Tamura et al., 2011) using the neighborjoining method. The reliability of each tree node was evaluated through bootstrap analysis with 1000 repetitions. The transmembrane domains and topology of the nAChR subunits were predicted by TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Finally, the identification of ORFs was conducted using ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/).

Tissue and developmental expression analysis of nAChR subunits To define the tissue-specific expression profiles of the nAChR subunits, total RNA was extracted from 10 different tissues in 50 fifth-instar larvae using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The tissues included the brain, subesophageal ganglion, thoracic ganglion, abdominal ganglion, hemocytes, fat body, foregut, midgut, hindgut and Malpighian tubules. For hemocyte collection, the fifth-instar larvae were surface-sterilized with 75% ethanol and total hemolymph was collected with a 20 μL sterilized pipette by cutting the proleg, followed by centrifugation at 200× g for 10 min at 4°C to collect the hemocyte precipitate (Wu et al., 2013b). Other tissues were dissected on ice from the fifth-instar larvae. To determine the developmental expression profile of the nAChR subunits, total RNA was extracted from eight different developmental stages: eggs, larvae (first-, second-, third-, fourth and fifth-instar), pupae and adults. For each RNA sample, 1 μg of total RNA was used to synthesize cDNA with TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, Beijing, China) for RT-PCR and quantitative real-time PCR. Specific primers for the RT-PCR and quantitative real-time PCR analyses were designed using Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/) (Tables S2 and S3). RT-PCR was performed in a 50 μL reaction containing 5 μL of 10× TaKaRa Ex Taq buffer, 0.5 μL of TaKaRa Ex Taq (TaKaRa, Kusatsu, Japan), 1 μL of cDNA template, 4 μL of dNTP mixture, 2 μL of each primer (10 μmol/L) and 35.5 μL of sterile H2 O. The PCR cycling parameters were as follows: 94°C for 3 min, followed by 40 cycles of 98°C for 10 s, 60°C for 30 s and 72°C for 1 min, with a final extension for 10 min at 72°C. The RT-PCR products were separated in 1.5% agarose gels stained with ethidium bromide. Each RT-PCR assay was performed with three biological replicates. Quantitative real-time PCR was conducted using the CFX ConnectTM C 2016

Real-Time Detection System (Bio-Rad, Hercules, CA, USA). The endogenous elongation factor 1 alpha (EF-1) gene was used to normalize the relative expression levels of the target genes (Wu et al., 2012). Quantitative realtime PCR was performed in 25 μL reactions containing R Premix Ex TaqTM II (Tli RNaseH 12.5 μL of SYBR Plus) (TaKaRa, Kusatsu, Japan), 1 μL of each primer (10 μmol/L), 5 μL of cDNA template, and 5.5 μL of sterile H2 O. The quantitative real-time PCR program was as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The PCR products were further used to measure the dissociation curves. Three independent biological replicates were performed to ensure reliability and reproducibility. Quantitative real-time PCR data analysis Relative quantification was performed via the comparative 2−CT method (Livak & Schmittgen, 2001). Before quantitative real-time PCR, we carried out some experiments to validate the efficiency of each primer by constructing a standard curve. Briefly, five 10× serial dilutions of cDNA from each sample were amplified using either the target or reference primers. For each serial dilution, mean CT was calculated and plotted against the log (dilution multiple). All data were normalized to the expression levels of the reference gene EF-1 in the same individual sample. For the analysis of relative expression levels in different tissues, the lowest expression level was taken as the calibrator. For the analysis of relative expression levels at different developmental stages, the expression level in eggs was employed as the calibrator. For the analysis of relative transcript abundances in the central nervous system, the nAChR subunit with the lowest expression level was taken as the calibrator. All results are indicated as the average of the expression levels in three independent biological replicates. The data on relative expression levels were analyzed through one-way analysis of variance (ANOVA), followed by a Tukey’s Honestly Significant Difference (HSD) test when significant differences were tested. All statistical analyses were run in the Data Processing System (DPS) package (Version 9.5) (Tang & Zhang, 2013). Results and Discussion Identification of nAChR subunit genes in C. suppressalis We searched C. suppressalis transcriptome data for nAChR subunit genes via local BLAST analysis, and their identities were further screened through a BLASTX



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Fig. 1 Alignment of Chilo suppressalis nAChR subunit protein sequences. Drosophila melanogaster α1 (Dmα1) is included for comparison. The dotted lines indicate N-terminal signal peptides. The positions of the loops (LpA-F) involved in ligand binding and the transmembrane motifs (TM1–TM4) forming the ion channel are indicated. The sites of the cysteine residues involved in cys-loop are marked with red asterisks and the vicinal cysteine residues characteristic of α-type subunits are boxed in red. The GEK motif associated with cation selectivity is indicated with inverted red triangles. Putative N-glycosylation sites are boxed. Potential protein kinase C phosphorylation and casein kinase II phosphorylation sites are indicated with dotted boxes, and potential tyrosine kinase phosphorylation sites are circled. Cyclic adenosine monophosphate (cAMP)- and cyclic guanine monophosphate (cGMP)-dependent protein kinase phosphorylation sites are underlined.

search of the NCBI database (Table S4), with 12 putative nAChR subunits being confirmed. Using RACE and RT-PCR approaches, the complete ORFs were obtained, and the full length of all 12 nAChR cDNA sequences was also validated. Multiple sequence alignment of the encoded proteins indicated that the C. suppres-

salis nAChR putative subunits exhibit typical features of the members of the cys-loop ligand-gated ion channel superfamily. These features include an N-terminal signal peptide, an extracellular N-terminal domain containing highly conserved amino acid residues in six loops (loops A–F) that are associated with ACh binding, a


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Table 1 nAChR subunits of eight insect species, including Chilo suppressalis (Cs), Bombyx mori (Bm), Drosophila melanogaster (Dm), Anopheles gambiae (Ag), Apis mellifera (Am), Nasonia vitripennis (Nv), Tribolium castaneum (Tc) and Acyrthosiphon pisum (Ap). nAChR subunits Name Cs Dα1 group Dα2 group Dα3 group Dα4 group Dα5–7 group Dβ1 group Dβ2 group Divergent nAChRs

α-type β-type Total

Bm

Dm

Ag

Am

Nv

Tc

Ap

α1 α2 α3 α4 α5, α6, α7 β1 α8

α1 α2 α3 α4 α5, α6, α7 β1 α8

α1 α2 α3 α4 α5, α6, α7 β1 β2

α1 α2 α3 α4 α5, α6, α7 β1 α8

α1 α2 α3 α4 α5, α6, α7 β1 α8

α1 α2 α3 α4 α5, α6, α7

α1 α2 α3 α4 α6, α7

α9, β2, β3

α9, β2, β3

β3

α9

α9, β2

α9, α10, β2

9 3 12

7 3 10

9 1 10

9 2 11

α9, α10, α11, α12, β2, β3, β4 12 4 16

α1 α2 α3 α4 α5, α6, α7 β1 α8, α11 α9, α10

11 1 12

9 2 11

9 3 12

dicysteine loop with two disulphide bond-forming cysteines separated by 13 residues, four transmembrane domains (TM1–TM4) in the C-terminal region that are associated with an ion channel forming, and a highly variable intracellular loop between TM3 and TM4 harboring phosphorylation sites. Among the 12 putative nAChR subunits identified in C. suppressalis, nine were designated as α-type subunits due to the presence of two vicinal cysteine residues that are necessary for ACh binding (Kao & Karlin, 1986) located in the loop C, and the other three were defined as β-type subunits due to lacking the adjacent residues (Fig. 1). The sequences reported in the present study have been submitted to GenBank with the following accession numbers: Csα1 (KP711043), Csα2 (KP711044), Csα3 (KP711045), Csα4 (KP711046), Csα5 (KP711046), Csα6 (KP711048), Csα6 variant 2 (KP711055), Csα7 (KP711049), Csα7 variant 2 (KP711056), Csα8 (KP711050), Csα8 variant 2 (KP711057), Csα9 (KP711051), Csβ1 (KP711052), Csβ2 (KP711053) and Csβ3 (KP711054). Comparison of C. suppressalis nAChR subunits with those identified in other insects The genomes of D. melanogaster, A. gambiae, A. mellifera, N. vitripennis, T. castaneum, A.pisum and B.

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β1 α8

β1 α8

mori have been completely sequenced, and nAChR subunit gene families have been subsequently identified, including seven α-type and three β-type subunits for D. melanogaster (Sattelle et al., 2005), nine α and one β for A. gambiae (Jones et al., 2005), nine α and two β for A. mellifera (Jones et al., 2006), 12 α and four β for N. vitripennis (Jones et al., 2010), 11 α and one β for T. castaneum (Jones & Sattelle, 2007), nine α and two β for A. pisum (Dale et al., 2010), and nine α and three β for B. mori (Shao et al., 2007) (Table 1). Similar to another lepidopteran species, the silkworm B. mori, 12 nAChR subunits were identified in C. suppressalis, including nine α-type subunits and three βtype subunits. There are 16 and 17 nAChR subunits in mammals and chicken, respectively (Millar & Denholm, 2007), whereas the largest known nAChR subunit gene family is that of the nematode Caenorhabditis elegans, with at least 27 subunits (Jones & Sattelle, 2004). Compared with vertebrates and the nematode, insects appear to exhibit a smaller nAChR gene family. Nevertheless, insects seem to increase the diversity of the functions of these subunits via RNA editing of transcripts and alternative splicing of exons (Sattelle et al., 2005; Jones et al., 2006). Most of the C. suppressalis nAChR subunits shared considerable sequence identity (up to 87%) with their



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Fig. 2 Phylogenetic relationships of 61 insect nAChR subunits from Drosophila melanogaster (Dm, 10), Nasonia vitripennis (Nv, 16), Tribolium castaneum (Tc, 11), Bombyx mori (Bm, 12) and Chilo suppressalis (Cs, 12). Neighbor-joining trees were constructed using MEGA 5 with 1000-fold bootstrap re-sampling. The numbers at the nodes of the branches represent the values of bootstrap support for each branch. The D. melanogaster FMRFamide receptor (DmFR) was used as an outgroup. The 12 C. suppressalis nAChR subunits are marked with triangles. The sequence accession numbers are shown in Table S5.


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Fig. 4 The tissue-specific expression patterns of the 12 nAChR subunits were evaluated through reverse transcription polymerase chain reaction in 10 tissues from Chilo suppressalis fifth-instar larvae, including the brain (Br), subesophageal ganglion (SOG), thoracic ganglion (TG), abdominal ganglion (AG), hemocytes (HC), fat body (FB), foregut (FG), midgut (MG), hindgut (HG) and Malpighian tubules (MT). The elongation factor 1 alpha (EF-1) gene was used as a reference gene.

Fig. 3 GEK motifs of all the divergent nAChR subunits of Drosophila melanogaster, Chilo suppressalis, Bombyx mori, Anopheles gambiae, Tribolium castaneum, Apis mellifera, Nasonia vitripennis and Acyrthosiphon pisum. The sequence accession numbers are shown in Table S5.

equivalents in D. melanogaster (Table S4). A phylogenetic tree was constructed using 61 nAChR subunit protein sequences from D. melanogaster, N. vitripennis, T. castaneum, B. mori and C. suppressalis (Fig. 2). Each of these nAChR gene families possess seven core groups of subunits which are highly conserved between different insect species (Table 1 and Fig. 2) (Jones et al., 2007). Similar to A. gambiae, A. mellifera, N. vitripennis, T. castaneum, A.pisum and B. mori, C. suppressalis also harbors subunit counterparts of Dα1–7, Dβ1 and Dβ2. Various insects possess the same number of core group subunits, with the exception of A. pisum, which lacks an α5 orthologue, and T. castaneum, which possesses an additional

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Dβ2 group subunit, most likely arising from a gene duplication event (Table 1) (Jones & Sattelle, 2007; Dale et al., 2010). Dα5, Dα6 and Dα7 have been assigned as a single group due to their high sequence homology to vertebrate α7 subunits (Jones et al., 2007). However, Csα5 shares a low sequence identity with Dα5, whereas Dα5 clusters nicely with nAChR α7 subunit in insects (Fig. 2). Thus D. melanogaster does not possess an insect α5 group orthologue, but two α7-like subunits. In C. suppressalis, α5 and α7 are difficult to identify. In B. mori, α7 was named because its N-terminal domain shared more identity with Dα7 and another was named α5 (Shao et al., 2007). We named C. suppressalis α5 and α7 following the nomenclature of B. mori because they share strong corresponding relationships. Interestingly, the orthologs of Dβ2 are all α subunits in seven other insect species (Table 1). Recent studies indicate that the putative Musca domestica ortholog of Dβ2 is also a non-α subunit (Scott et al., 2014), which suggests that it could be characteristic of the Brachycera suborder, and a change in the functional role of the subunits may have occurred (Jones & Sattelle, 2010).



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Fig. 5 The tissue-specific expression patterns of the 12 nAChR subunits were analyzed via quantitative real-time polymerase chain reaction in 10 tissues from Chilo suppressalis fifth-instar larvae, including the brain (Br), subesophageal ganglion (SOG), thoracic ganglion (TG), abdominal ganglion (AG), hemocytes (HC), fat body (FB), foregut (FG), midgut (MG), hindgut (HG) and Malpighian tubules (MT). The standard error is represented by the error bar, and the different letters above each bar denote significant differences (P < 0.05).

In addition, the analysis of eight nAChR subunit gene families suggested that the investigated insect species harbor at least one divergent nAChR subunit (Table 1), showing low sequence homology with all other known nAChR subunits (Fig. 2). Each of these eight insects exhibits a variable range of divergent subunits. For instance, both C. suppressalis and B. mori harbor three divergent subunits, including one α and two β subunits, whereas there are four α and three β subunits in N. vitripennis (Table 1). Although C. suppressalis and B. mori are both lepidopteran insects, the divergent subunits Csβ2 shows only 29% sequence identity with Bmβ2, and Csβ3 shows 33% identity with Bmβ3. Therefore, the divergent nAChR subunits are likely to play species-specific roles, and thus may be attractive distinctive targets for controlling insect pests without impacting beneficial insects (Jones & Sattelle, 2010). The divergent subunits exhibit exceedingly short intracellular regions between TM3 and TM4, in addition to showing low sequence homology, and all of the

divergent nAChR subunits of these eight insect species (except Dmβ3 and Tcα10) (Fig. 3) lack the highly conserved GEK motif upstream of TM2, which is associated with ion permeation and selectivity (Jensen et al., 2005).

Tissue expression profiles of nAChR subunits The tissue-specific expression patterns of C. suppressalis nAChR subunits were detected in 10 different tissues from the fifth-instar larvae, including the brain, subesophageal ganglion, thoracic ganglion, abdominal ganglion, hemocytes, fat body, foregut, midgut, hindgut and Malpighian tubules using RT-PCR and quantitative real-time PCR methods. Among the 12 nAChR subunits, Csα1, Csα2, Csα3, Csα4, Csα5, Csα6, Csα7, Csα8 and Csβ1 were highly expressed in the central nervous system, including the brain, subesophageal ganglion, thoracic


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Fig. 6 The developmental stage-specific expression patterns of the 12 nAChR subunits were analyzed via quantitative real-time polymerase chain reaction in eight stages, including the eggs, first-instar larvae (L1), second-instar larvae (L2), third-instar larvae (L3), fourth-instar larvae (L4), fifth-instar larvae (L5), pupae and adults. The standard error is represented by the error bar, and the different letters above each bar denote significant differences (P < 0.05).

ganglion and abdominal ganglion, showing the highest expression in the brain and subesophageal ganglion, whereas they were virtually undetectable in the hemocytes, fat body, foregut, midgut, hindgut and Malpighian tubules (Fig. 4 and Fig. 5). It is believed that nAChRs play a critical role in the central nervous system and the high enrichment of these nAChRs within the central nervous system has been beneficial for the exploitation of insecticides targeting these receptors (Song et al., 2009). Interestingly, Csα9, Csβ2 and Csβ3 were all expressed at the highest levels in the fat body, followed by the foregut, whereas they were all expressed at low levels in the central nervous system (Fig. 4 and Fig. 5). In the codling moth C. pomonella, the transcripts of α9, β2 and β3 were also detected in heads and bodies of larvae and pupae, whereas α9, β2 and β3 only appear in the bodies of adults (Martin & Garczynski, 2016). Thus, the functional roles of these highly expressed nAChRs in the fat body remain to be further investigated.

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Fig. 7 The relative transcript abundance of the 12 nAChR subunits in the central nervous system of Chilo suppressalis fifthinstar larvae was determined via quantitative real-time polymerase chain reaction. The standard error is represented by the error bar, and the different letters above each bar denote significant differences (P < 0.05).



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Fig. 8 Alignment of the amino acid sequences in loops D, E and F of the nicotinic acetylcholine receptor β1 subunits of Drosophila melanogaster (Dmβ1), Chilo suppressalis (Csβ1), Bomby mori (Bmβ1), Tribolium castaneum (Tcβ1), Apis mellifera (Amβ1), Locusta migratoria (Lmβ1), Nilaparvata lugens (Nlβ1), Pardosa pseudoannulata (Ppβ1), Ixodes scapularis (Isβ1) and Myzus persicae (Mpβ1). Fully conserved identical residues are shaded in black, whereas similar residues are shaded in grey. The red asterisks mark the residues (R81, V83, R135, N137, F190, W197 and K198 based on M. persicae nAChR β1 numbering) involved in imidacloprid selectivity. An R81T point mutation is strongly associated with neonicotinoid resistance in M. persicae. The sequence accession numbers are shown in Table S5.

Developmental expression profiles of nAChR subunits The developmental expression profiles of the 12 nAChR subunits of C. suppressalis were quantified in the eggs, five larval instars (first, second, third, fourth and fifth), pupae and adults. The quantitative real-time PCR results showed that the expression levels of the nAChR subunits varied between the developmental stages. Csα1, Csα2, Csα3, Csα4, Csα5, Csα6, Csα7, Csα8 and Csβ1 were significantly expressed in the first-instar larvae. For these nine nAChR subunits, their transcripts decreased gradually in larval stages, and then began to increase at the pupal stage (Fig. 6). Interestingly, the expression level of Csα9 was highest in the eggs and decreased gradually until the pupal stage. In addition, Csβ2 and Csβ3 both showed higher expression levels in the pupal and adult stages (Fig. 6). Our results indicated that the spatial and temporal expression profiles of the divergent nAChR subunits (Csα9, Csβ2 and Csβ3) appeared to be different from the seven core groups.

the 12 nAChR subunits of C. suppressalis was observed for Csβ1, followed by Csα1, Csα6, Csα7, Csα8, Csα3, Csα2, Csα4, Csα5, Csα9, Csβ2 and Csβ3 (Fig. 7). A previous study showed that β1 appears to be the most abundant non-α nAChR subunit in insects (Yao et al., 2008), and similarly β1 was found to be the most abundant nAChR subunit in C. suppressalis in our study. Protein sequence alignment of loops D, E and F of the nAChR β1 subunits of insects and other arthropods indicated that several amino acids are associated with neonicotinoid resistance. These residues (R81, V83, R135, N137, F190, W197 and K198 based on Myzus persicae nAChR β1 numbering) are involved in imidacloprid selectivity (Dermauw et al., 2012). The key role of position 81 in loop D was demonstrated by a previous study, suggesting that an R81T mutation was closely associated with neonicotinoid resistance in M. persicae (Fig. 8) (Bass et al., 2011, Puinean et al., 2013). Further functional investigation of nAChR subunits is required to identify key target-sites associated with resistance to a variety of insecticides and to illustrate the mode of action of insecticide resistance in C. suppressalis.

Relative transcript abundance of nAChR subunits in the central nervous system Acknowledgments The relative transcript abundance of the 12 nAChR subunits in the C. suppressalis central nervous system were determined via quantitative real-time PCR. Our results indicated that the highest expression level among

This work was supported by National Special Agricultural Research Projects for Public Welfare, China (201303017), National Science Fund for Innovative


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Research Groups of Biological Control (Grant 31321063) and National High-Tech R&D Program of China (863 Program, 2011AA10A204). The authors sincerely thank Shuang-Yang Wu, Pi-Hua Zhou, Fang Liu, Lu-Lu Gu and Gui-Xiang Gu for assistance in collecting and feeding the rice striped stem borer. Disclosure The authors declare that they have no conflicts of interest. References Adams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., Gocayne, J.D., Amanatides, P.G., Scherer, S.E., Li, P.W., Hoskins, R.A. and Galle, R.F. et al. (2000) The genome sequence of Drosophila melanogaster. Science, 287, 2185–2195. Albuquerque, E.X., Pereira, E.F.R., Alkondon, M. and Rogers, S.W. (2009) Mammalian nicotinic acetylcholine receptors: from structure to function. Physiological Reviews, 89, 73– 120. Bass, C., Puinean, A.M., Andrews, M., Cutler, P., Daniels, M., Elias, J., Paul, V.L., Crossthwaite, A.J., Denholm, I. and Field, L.M. et al. (2011) Mutation of a nicotinic acetylcholine receptor β subunit is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae. BMC Neuroscience, 12, 51. Chang, C., Cheng, X., Huang, X.Y. and Dai, S.M. (2014) Amino acid substitutions of acetylcholinesterase associated with carbofuran resistance in Chilo suppressalis. Pest Management Science, 70, 1930–1935. Collin, C., Hauser, F., De Valdivia, E.G., Li, S., Reisenberger, J., Carlsen, E.M.M., Khan, Z., Hansen, N.O., Puhm, F., Sondergaard, L. and Niemiec, J. et al. (2013) Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods. Cellular and Molecular Life Sciences, 70, 3231– 3242. Dacher, M., Lagarrigue, A. and Gauthier, M. (2005) Antennal tactile learning in the honeybee: effect of nicotinic antagonists on memory dynamics. Neuroscience, 130, 37–50. Dale, R.P., Jones, A.K., Tamborindeguy, C., Davies, T.G., Amey, J.S., Williamson, S., Wolstenholme, A., Field, L.M., Williamson, M.S., Walsh, T.K. and Sattelle, D.B. (2010) Identification of ion channel genes in the Acyrthosiphon pisum genome. Insect Molecular Biology, 19 Suppl 2, 141–153. Dermauw, W., Ilias, A., Riga, M., Tsagkarakou, A., Grbic, M., Tirry, L., van Leeuwen, T. and Vontas, J. (2012) The cys-loop ligand-gated ion channel gene family of Tetranychus urticae: Implications for acaricide toxicology and a novel mutation associated with abamectin resistance. Insect Biochemistry and Molecular Biology, 42, 455–465. C 2016

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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1 Primers used for the cloning of nicotinic acetylcholine receptor genes in C. suppressalis. Table S2 Primers used for the RT-PCR analysis of nicotinic acetylcholine receptor genes in C. suppressalis. Table S3 Primers used for the quantitative real-time PCR analysis of the expression levels of nicotinic acetylcholine receptor genes in C. suppressalis. Table S4 Percentage of identity between putative C. suppressalis and D. melanogaster nAChR subunits. Table S5 The accession numbers of the sequences used in this study.
