Insect Molecular Biology

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Insect Molecular Biology (2014)

doi: 10.1111/imb.12143

Two subclasses of odorant-binding proteins in Spodoptera exigua display structural conservation and functional divergence

N.-Y. Liu*†1, F. Yang*1, K. Yang*, P. He*, X.-H. Niu*, W. Xu†, A. Anderson† and S.-L. Dong* *Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing, China; and †CSIRO Ecosystem Sciences, Canberra, ACT, Australia Abstract Although many studies on lepidopteran pheromonebinding proteins (PBPs)/ general odorant-binding proteins (GOBPs) have been reported, the functional differentiation within and between the two odorantbinding protein (OBP) subclasses is still elusive. Here we conducted a comparative study on three SexiPBPs and two SexiGOBPs in Spodoptera exigua. Results showed that all five SexiPBP/GOBP genes have the same intron numbers and conserved exon/intron splice sites. Reverse transcription PCR results showed that these five SexiPBP/GOBPs were primarily expressed in antennae of both sexes and some were also detected in other tissues. Further, quantitative real-time PCR showed that five SexiPBP/GOBPs had different sex-biased expression patterns, with PBP1 being highly male-biased (5.96-fold difference) and PBP3 slightly female-biased (2.43-fold difference), while PBP2 and two GOBPs were approximately sex-equivalent (the absolute value < 1.90-fold difference). Binding assays showed that all three SexiPBPs could bind all six sex pheromone components, but SexiPBP1 had much higher affinities [dissociation constant (Ki) < 1.10 μM] than did the other two SexiPBPs (Ki > 1.20 μM). Very intriguingly,

SexiGOBP2 displayed even stronger binding to five sex pheromone components (Ki < 0.40 μM) than SexiPBP1. In contrast, SexiGOBP1 only exhibited weak binding to three alcohol-pheromone components. Similar results were obtained for tested pheromone analogues. In addition, each of SexiPBP/GOBPs selectively bound some plant odorants with considerable affinities (Ki < 10.0 μM). Taken together, of the three SexiPBPs, SexiPBP1 may play the most important role in female sex pheromone reception, and additionally all three SexiPBPs can detect some plant odorants, while SexiGOBP2 may be involved in the detection of female sex pheromones in addition to plant odorants. The results strongly suggest functional differentiation within and between the two OBP sub-classes. Keywords: Spodoptera exigua, odorant-binding protein, gene structure, expression pattern, fluorescence binding assay.

Introduction

These authors contributed equally to this work.

In insect moths, olfaction plays a central role in processing chemical signals from the external environment, leading to the detection of food sources, reproductive partners, oviposition sites, hosts and prey or predators (Hallem & Carlson, 2006). The odorant-binding proteins (OBPs) are small molecular weight (15–17 kDa) and water-soluble proteins present in the sensillar lymph of olfactory neurons in extremely high concentrations (up to ∼10 mM in Antheraea polyphemus; Klein, 1987). The functions of OBPs are postulated to be involved in the uptake, binding and transport of odorant molecules (Leal, 2013), as well as odorant recognition (Du & Prestwich, 1995) and receptor activation (Grosse-Wilde et al., 2007). In Lepidoptera, pheromone-binding proteins (PBPs) and general odorant-binding proteins (GOBPs) consist of two specific subclasses of PBP/GOBPs and are distinguished from other lepidopteran OBPs and those of other insect

© 2014 The Royal Entomological Society

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Correspondence: Shuang-Lin Dong, Department of Entomology, College of Plant Protection, Nanjing Agricultural University, No. 1 Weigang, Xuanwu District, Nanjing 210095, China. Tel./fax: +86 25 84399062; e-mail: [email protected] 1

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orders (Vogt et al., 2002). The former is thought to be involved in the detection of sex pheromone produced by females (Vogt & Riddiford, 1981), and the latter is proposed to be associated with the reception of general odorant released by food and host plants (Vogt et al., 1991a). The first moth PBP was identified from the silkworm A. polyphemus (Vogt & Riddiford, 1981). Subsequently, two or more PBPs have been found within a single noctuid species, such as Agrotis ipsilon (Picimbon et al., 1997; Gu et al., 2013), Helicoverpa armigera (Zhang et al., 2011), Spodoptera litura (Liu et al., 2013), Spodoptera littoralis (Legeai et al., 2011), Sesamia inferens (Jin et al., 2014) and Sesamia nonagrioides (Glaser et al., 2013). Notably, each subgroup of the PBP is phylogenetically grouped into three distinct clades (Picimbon, 2003), suggesting different functional roles in sex pheromone communication. Additionally, it is well known that the sex pheromone of these noctuid species is a blend of multiple components produced by female pheromone glands. For example, in H. armigera, 12 components of female sex pheromone have been isolated and identified (EI-Sayed, 2014), and similarly in A. ipsilon (Picimbon et al., 1997), Helicoverpa assulta (Cork et al., 1992) and Spodoptera exigua (Dong & Du, 2002; Acín et al., 2010), at least four different components have been reported so far. Considering this nature of noctuid multiple PBPs and multiple sex pheromones, it is hypothesized that each subgroup of the PBP might be narrowly tuned to a specific component(s) in pheromone recognition. Indeed, in Lymantria dispar (Plettner et al., 2000), A. polyphemus (Mohl et al., 2002) and A. ipsilon (Gu et al., 2013) studies showed that each subgroup of the PBP could selectively bind the specific component(s) of sex pheromones; however, different results were presented in Mamestra brassicae (Campanacci et al., 2001), H. armigera and H. assulta (Guo et al., 2012) as well as S. litura (Liu et al., 2012a, 2013), in which the PBPs did not show an obvious binding specificity to the sex pheromones. Moreover, some similar results were also obtained in non-noctuid species including European and Asian corn borer (Willett & Harrison, 1999). The data from gene expression studies using reverse transcription (RT)-PCR and quantitative real-time (q)PCR, immunolocalization and in situ hybridization further showed that moth PBPs have diversified tissue and sensilla expression patterns. In Lepidoptera, moth PBPs were primarily expressed in adult antennae, but were also detected in non-antennal tissues such as wing, proboscis and labial palp (Guo et al., 2012; Gu et al., 2013; Sun et al., 2013). In addition, in the noctuid moth Heliothis virescens and Autographa gamma, the PBPs were labelled in basiconica sensilla and male long trichoid sensilla (Zhang et al., 2001). In non-noctuid moths,

Manduca sexta (Nardi et al., 2003) and Bombyx mori (Forstner et al., 2006) PBP2 and PBP3 were expressed in short trichoid sensilla that are different from pheromonesensitive long trichoid sensilla expressed the PBP1. These results further suggest functional divergence among PBPs. Although so far three groups of the PBPs have been found in noctuid moths, in most of these species the number of PBPs is less than that of sex pheromones. This possibly suggests that other groups of OBPs may be also involved in the binding and transport of the sex pheromone molecules. The GOBPs (including GOBP1 and GOBP2) are other crucial members of the PBP/GOBP groups in Noctuidae and are also possibly responsible for carrying the sex pheromones. Although these GOBPs were previously reported to be detected only in basiconica sensilla associated with general odorant reception (Vogt et al., 1991a; Steinbrecht et al., 1995), a different and diverse expression has since been found in several noctuid moths. For example, in M. brassicae MbraGOBP2 expression was restricted to the long trichoid sensilla that house the pheromone-sensitive neurons (Jacquin-Joly et al., 2000). In H. virescens, H. armigera, A. gamma and S. littoralis, the GOBP2 was detected in trichoid sensilla (Zhang et al., 2001) in addition to basiconica sensilla. For non-noctuid moths, the GOBP1s were detected in basiconica sensilla and pheromone-sensitive trichoid sensilla in M. sexta (Nardi et al., 2003) and B. mori (Maida et al., 2005). The beet armyworm, S. exigua Hübner (Lepidoptera: Noctuidae), is a serious pest of various agricultural crops in Asia and North America (C.A.B., 1972). Its sex pheromone has been identified as a four-component blend of Z9-14:Ac, Z9,E12-14Ac, Z9-14:OH and Z9,E12-14:OH in Chinese populations (Dong & Du, 2002) and a sixcomponent blend with two additional components of Z1116:Ac and Z11-16:OH in Spanish populations (Acín et al., 2010). Two PBPs (SexiPBP1 and SexiPBP2; Xiu & Dong, 2007) and one GOBP (SexiGOBP2; Wang et al., 2001) have previously been identified from S. exigua adult antennae. In the present study, we identified two other novel OBP full-length genes from male antennae, namely SexiPBP3 and SexiGOBP1 based on National Center for Biotechnology Information (NCBI) BLAST results and the phylogenetic tree, leading to a total of three SexiPBPs and two SexiGOBPs in this species. With the availability of these five genes, we further addressed the issue of whether these PBPs and GOBPs in S. exigua have been functionally differentiated into the cross detection between sex pheromone and/or general odorant. The results provide new insight into the molecular mechanisms underlying sex pheromone and general odorant recognition in lepidopteran PBPs and GOBPs. © 2014 The Royal Entomological Society

Odorant-binding proteins in Spodoptera exigua Results Identification and characterization of two Spodoptera exigua OBPs By a bioinformatics-based approach we identified fragments of two OBP genes from S. exigua transcriptome. Further, the cDNA full-length sequences of two OBP genes were cloned by rapid amplification of cDNA ends, and named as SexiPBP3 (GenBank accession number: GU082320) and SexiGOBP1 (GenBank accession number: GU082319) based on a NCBI BLAST hit and the phylogenetic tree (Fig. 1A). Both genes have the conserved motifs of lepidopteran OBPs, including six conserved cysteine residues and a predicted signal sequence (Fig. S1). The cDNA sequence of candidate SexiPBP3 is 564 bp in length and contains an open reading frame (ORF) of 495 nucleotides that encodes 165 amino acids with a predicted signal sequence of 22 amino acids (Fig. S1). SexiPBP3 shared ∼87 to 89% identity with other noctuid PBP3s reported, including A. ipsilon, H. armigera, H. assulta, S. inferens, S. littoralis, S. litura and S. nonagrioides. A relatively low identity was observed to other two SexiPBPs, with 51% for PBP1 and 45% for PBP2 (Fig. 1A). Using genomic DNA as the template, we A

Figure 1. Neighbour-Joining tree of Spodoptera exigua odorant-binding proteins (OBPs) with other noctuid pheromone-binding proteins (PBPs)/ general odorant-binding proteins (GOBPs) (A) and gene structures of five S. exigua PBP/GOBPs (B). The tree was constructed with MEGA 6.06 using the Jones-Taylor-Thornton model with 1000 bootstrap replicates. Five SexiPBP/GOBPs are highlighted in red. The exon/intron splice sites were analysed using a GeneWise program based on the genomic DNA sequences of SexiPBP/GOBPs. All gene accession numbers are available in Table S2. Aips: Agrotis ipsilon, Aseg: Agrotis segetum, Harm: Helicoverpa armigera, Hass: Helicoverpa assulta, Hvir: Heliothis virescens, Hviri: Heliothis viriplaca, Hzea: Helicoverpa zea, Mbra: Mamestra brassicae, Msep: Mythimna separata, Sexi: Spodoptera exigua, Sinf: Sesamia inferens, Slitt: Spodoptera littoralis, Slit: Spodoptera litura, Snon: Sesamia nonagrioides.

© 2014 The Royal Entomological Society

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identified the genomic DNA sequence of SexiPBP3 (GenBank accession number: KF840410), which contains two introns of Intron1 (1824 bp) and Intron2 (75 bp). The first intron is located between Glu44 and Leu45 and the second is present inside the codon for Gly105. The boundaries of both introns have a typical GT–AG structure (Fig. S1). The cDNA sequence of candidate SexiGOBP1 is 876 bp and contains an ORF of 495 nucleotides that encodes 165 amino acids with a signal peptide of 19 amino acids (Fig. S1). SexiGOBP1 showed a high amino acid identity of >89% to GOBP1 from other noctuid species, with the highest identity of 94% to GOBP1 in S. littoralis (Fig. 1A). The genomic DNA sequence of SexiGOBP1 (GenBank accession number: KF840411) also contains two introns of Intron1 (174 bp) and Intron2 (76 bp) with the typical GT–AG structure. The first intron is located between Glu41 and Ser42, and the second is present inside the codon for Gly102 (Fig. S1).

Phylogenetic analysis and gene structure of five Spodoptera exigua PBP/GOBPs To analyse the phylogenetic relationships of SexiPBP/ GOBPs with other noctuid PBP/GOBPs, we constructed

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a neighbour-joining tree. Results show that five SexiPBP/GOBPs are clearly distinguished into two large clades; namely, PBP clade and GOBP clade. The PBP clade is further divided into three distinct groups (Group1, Group2, and Group3) and the GOBP clade is clustered into two conserved groups (Group4 and Group5) (Fig. 1A). Based on genomic DNA sequences of five SexiPBP/ GOBPs, we further analysed their exon and intron structural characteristics. All these genes have three exons (Exon1, Exon2 and Exon3) and two introns (Intron1 and Intron2), with conserved exon/intron splice sites. Intron1 is inserted between two amino acids and Intron2 is present within a triplet codon. Compared with exons, the length of introns is diverse among different PBP and GOBP genes or Intron1 and Intron2 (Fig. 1B). In addition, exon sequences exhibited much higher identities than intron sequences (data not shown).

Tissue- and sex-specific expression pattern of five Spodoptera exigua PBP/GOBPs To determine the global expression patterns of five SexiPBP/GOBPs in different tissues of both sexes, we carried out RT-PCR. The results showed that all five SexiPBP/GOBPs were primarily expressed in the antennae of both sexes. In addition, SexiPBP1 expression was weakly detected in male proboscises and heads; SexiPBP2 was also expressed in the proboscises, heads and legs of both sexes, as well as the male abdomen. In comparison, two SexiGOBPs exhibited a broader expression pattern, including female and male proboscises, heads, legs and male wing as well as female pheromone gland (Fig. 2A). Further, sex-biased expression of all these genes in female and male antennae was determined by qPCR. These five SexiPBP/GOBP genes had different sex-biased expression patterns. SexiPBP1 was highly male-biased, with the transcription level in males being 5.96-fold higher than that in females. By contrast, SexiPBP3 was slightly female-biased with the mRNA amount being 2.43-fold higher in females than that in males. SexiPBP2, compared with SexiPBP1 and SexiPBP3, exhibited a more similar expression between females and males with a 1.77-fold difference. Among these three SexiPBPs, the relative ratios of PBP1 : PBP2 : PBP3 are 16.5:1.5:1.0 in males and 1.1:1.1:1.0 in females (Fig. 2B). In addition, two SexiGOBPs expression did not show an obvious preference in antennae of both sexes, with a 1.81-fold difference for GOBP1 between males and females as well as a 1.07-fold difference for GOBP2 between females and males (Fig. 2C).

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Figure 2. Sex- and tissue-specific expression of five Spodoptera exigua pheromone-binding proteins (PBPs)/ general odorant-binding proteins (GOBPs). (A) Expression patterns of five SexiPBP/GOBP genes in different tissues of both sexes. Negative control (N) was performed using sterile water as the template. S. exigua GAPDH gene was used as quality and quantity control for all cDNA templates. A, antenna; P, proboscis; H, head without antenna and proboscis; T, thorax; Ab, abdomen (female abdomen without pheromone gland); L, leg; W, wing and Pg, pheromone gland. (B) Relative transcription levels of three SexiPBP genes in antennae of both sexes. (C) Relative transcription levels of two SexiGOBP genes in antennae of both sexes. Bars represent standard errors of three independent biological pools, with three replicates of each pool.

Temporal expression of three Spodoptera exigua PBPs The transcription of three SexiPBP genes was quantified by qPCR in male antennae on different days. As a result, the transcription of all three SexiPBPs was detectable 2 days before moth emergence, although the expression levels were very low. After emergence, the transcription levels climbed rapidly and reached a peak by day 3. Compared with the third day after emergence, the expression levels of SexiPBP1 and SexiPBP3 decreased by day 4, and then stayed stable until day 6; however, the expression of SexiPBP2 showed a slow reduction until day 5 and then increased by the day 6 (Fig. 3A). Because of the higher expression of three SexiPBPs on day 3, we further measured their expression levels at 14 different times on day 3, including the scotophase and photophase periods. Results showed that all three SexiPBPs generally displayed consistent expression during the entire day, although a small expression peak was observed between the late scotophase and early photophase for SexiPBP1 and SexiPBP2 (Fig. 3B). © 2014 The Royal Entomological Society

Odorant-binding proteins in Spodoptera exigua A

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Figure 3. Temporal expression of three Spodoptera exigua pheromone-binding proteins (PBPs) in male antenna. (A) Daily expression profiles of SexiPBPs before and after moth emergence. The antennae were collected between the 8th and 9th hours of scotophase. Numbers on the x-axis represent the days of eclosion with ‘-2 and 0′ as two days before moth eclosion and that day of eclosion, respectively. (B) Hourly expression profiles of SexiPBPs from 3-day-old male antennae during scotophase (S) and photophase (P). S1 to S10 mean the 1st (23:00 h) to 10th (8:00 h) hours after scotophase. P1 to P13 mean the 1st (9:00 h) to 13th (21:00 h) hours after photophase. Data were analysed with a one-way ANOVA followed by Tukey’s test (P < 0.05). Fisher’s least-significant difference test was used for multiple comparisons. Bars represent standard errors of three independent biological pools.

Bacterial expression and purification of five Spodoptera exigua PBP/GOBPs The proteins were expressed in a bacterial system, and showed a high yield with 10–30 mg/l culture (Fig. 4). All these proteins were present in inclusion bodies, and thus solubilization was carried out by denaturation and renaturation according to previously reported protocols (Ban et al., 2003; Calvello et al., 2003; Liu et al., 2012a). Purification was performed by an affinity chromatography XK 16/20 column, and an apparent protein band of ∼22 to 23 kDa with His-tags was observed (Fig. 4). To avoid the possible effects of His-tags on subsequent experiments, the His-tags were removed by digestion with recombinant enterokinase. After digestion, the protein was re-purified by the affinity chromatographic column and finally an expected target band of ∼16 to 17 kDa was obtained (Fig. 4). Ligand binding assays of five Spodoptera exigua PBP/GOBPs To investigate and compare the binding specificity of five SexiPBP/GOBPs to sex pheromone and non-pheromone © 2014 The Royal Entomological Society

ligands, a fluorescence competitive binding assay was used. First, binding affinities of N-phenyl-1-naphthylamine (1-NPN) to five SexiPBP/GOBPs were measured by titrating increasing concentrations of 1-NPN. All these SexiOBP proteins could bind 1-NPN with a micromole range of dissociation constants (PBP1: 3.42 ± 0.29 μM; PBP2: 5.52 ± 0.40 μM; PBP3: 5.75 ± 0.21 μM; GOBP1: 8.90 ± 0.77 μM; and GOBP2: 2.54 ± 0.20 μM) (Fig. 5A). Next, competitive binding assays of five SexiPBP/ GOBPs to different odorants were carried out using 1-NPN as the fluorescent reporter. In general, all sex pheromones and most of their analogues could bind strongly to SexiPBP1 and SexiGOBP2, with much higher affinities than the other three SexiPBP/GOBPs. Interestingly, the binding affinity of SexiGOBP2 to the major sex pheromone Z9,E12-14:Ac was even stronger than that of SexiPBP1, with a 4.76-fold difference in dissociation constant (Ki) values. Similar results between SexiGOBP2 and SexiPBP1 were also observed for Z9-14:Ac and the other three alcohol-pheromones; however, the minor sex pheromone Z11-16:Ac exhibited much weaker binding to SexiGOBP2, relative to SexiPBP1 (Fig. 5B, C and Table 1).

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Expression

Purification

Of tested plant odorants, linoleic acid and β-ionone were the best ligands of five SexiPBP/GOBPs, and in particular β-ionone showed a stronger binding to all three SexiPBPs (Ki < 10.0 μM) than two SexiGOBPs (Ki > 11.0 μM). Notably, all tested carboxylic acids exhibited relatively strong binding to SexiPBP1 and SexiPBP2 (0.40 μM < Ki < 13.0 μM). Farnesol also exhibited a strong binding affinity to SexiPBP1 (Ki = 5.19 μM), and some plant odorants, including nerolidol, farnesol, oleic acid and 2-pentadecanone, were all good ligands of SexiGOBP2 (Ki < 10.0 μM). For SexiPBP3 and SexiGOBP1, most plant odorants proved to be poor ligands (Fig. 5D–F and Table 1). We found that the ligand structural features may have affected the binding with SexiPBP/GOBPs. For the C14-16 linear acetates, SexiPBP1 showed much stronger binding to the ligands with a double bond in the ninth position, such as Z7-14:Ac, Z9-14:Ac and Z11-14:Ac, and 16:Ac, Z9-16Ac and Z11-16:Ac. In addition, SexiGOBP2 could strongly bind the linear ligands with the double bond(s), but not those ligands without the double bond, including 16:Ac, Z9-16Ac and Z11-16:Ac, and stearic acid, oleic acid and linoleic acid (Fig. 5 and Table 1). Effect of pH on binding affinities of five Spodoptera exigua PBP/GOBPs to Z9,E12-14:Ac The major sex pheromone Z9,E12-14:Ac was selected to measure the effect of pH on binding affinities. Results showed that the binding (indicated by the fluorescence

Figure 4. Expression and purification of five Spodoptera exigua pheromone-binding proteins (PBPs)/ general odorant-binding proteins (GOBPs). Upper panel: expression of five SexiPBP/GOBPs in a bacterial system. A expression vector that was not inserted the target gene was used as control (lane 1 and 2: before and after IPTG induction, respectively). Crude bacterial extracts before (lane 3) and after (lane 4) 0.2 mM IPTG induction were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Lower panel: purified protein samples with His-tag (lane 2) and without His-tag (lane 1). The His-tag was removed by recombinant enterokinase (lane 3) and target proteins are indicated by arrows.

displacement percent) of SexiPBP1 to this component was heavily affected by pH, with a reduction in fluorescence displacement from 61.4% (pH 7.0) to 11.5% (pH 5.0) at 4.0 μM, while the binding of SexiPBP3 appeared to be slightly affected and the binding of the other three SexiOBPs was moderately affected (Fig. 6). Since the binding of SexiPBP1 to Z9,E12-14:Ac showed a stronger pH-dependent change than the other four OBPs, we further examined the effects of pH on SexiPBP1 conformation and its binding to 1-NPN under nine different pH conditions, ranging from 4.0 to 9.0. As a result, SexiPBP1 intrinsic fluorescence and SexiPBP1/1NPN binding exhibited a very similar pH-dependent pattern, with a slight increase of fluorescence intensity from pH 4.0 to 5.0, a steep increase from pH 5.0 to 6.5, and then generally remaining stable from pH 6.5 to 9.0 (Fig. S2). Discussion Identification of two candidate Spodoptera exigua OBP genes To date, PBP3 orthologues have been found in eight Noctuidae (Yang et al., 2010; Legeai et al., 2011; Guo et al., 2012; Jacquin-Joly et al., 2012; Zhang et al., 2012; Glaser et al., 2013; Gu et al., 2013; Jin et al., 2014), including SexiPBP3 reported in the present study; however, the number of noctuid PBP3s reported is lower than that of PBP1 (11) and PBP2 (14), probably because of its lower expression level. Our qPCR results showed that the © 2014 The Royal Entomological Society

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Figure 5. Binding of tested ligands to five Spodoptera exigua pheromone-binding proteins (PBPs)/ general odorant-binding proteins (GOBPs). (A) Binding curves of N-phenyl-1-naphthylamine (1-NPN) to recombinant SexiPBP/GOBPs and relative Scatchard plots. A 2 μM solution of each protein in Tris-buffer (pH 7.4) was titrated with 1 mM 1-NPN in methanol to final concentrations of 2–20 μM. Dissociation constants (N = 3) were PBP1: 3.42 μM (SEM 0.29); PBP2: 5.52 μM (SEM 0.40); PBP3: 5.75 μM (SEM 0.21); GOBP1: 8.90 μM (SEM 0.77); GOBP2: 2.54 μM (SEM 0.20). (B–F) Comparative binding curves of five SexiPBP/GOBPs to sex pheromones (B), pheromone analogues (C), carboxylic acids (D), alcohols (E) and other odorants (F).

transcription level of SexiPBP3 is only approximately onetenth that of SexiPBP1 and half that of SexiPBP2 in male antenna. Since noctuid PBPs were clearly divided into three distinct conserved subgroups, we suppose that all noctuid species have three different PBPs. In S. exigua, two PBPs (SexiPBP1 and SexiPBP2; Xiu & Dong, 2007) and one GOBP (SexiGOBP2; Wang et al., 2001) have been previously reported. In the present study, we identified two more OBPs (SexiPBP3 and SexiGOBP1) and their genomic DNA sequences, making all three PBPs and two GOBPs available in S. exigua. This allows us to conduct comparative studies on gene structures and functions of all five PBP/GOBPs in S. exigua, and provides more information to address functional differentiation of PBPs and/or GOBPs in S. exigua as well as other lepidopteran species.

Phylogenetic analysis and gene structure of Spodoptera exigua PBP/GOBPs In S. exigua, five SexiPBP/GOBPs are well clustered into five distinct groups, suggesting their different roles in odorant recognition. It is noted that the nomenclature of © 2014 The Royal Entomological Society

PBPs in Group1 and Group2 is not consistently used by authors. In Group1, members in some species were named PBP1, and others PBP2. Similarly in Group2, some were named PBP1 and others PBP2. In the previous study (Xiu & Dong, 2007), SexiPBP of Group1 was designated as PBP2, and SexiPBP in Group2 was named PBP1, according to the previous nomenclature used in M. brassicae (Nagnan-Le Meillour et al., 1996; Maibeche-Coisne et al., 1998) and S. nonagrioide (de Santis et al., 2006). Lepidopteran PBP/GOBPs comprise two distinguishable and specific OBP subclasses from other OBPs in Lepidoptera and other insect orders, with respect to gene structures containing exclusively two introns at conserved sites (Vogt et al., 2002). In contrast, other OBPs from Lepidoptera (Gong et al., 2009) and other orders (Zhou et al., 2010) generally have a variety of intron numbers and exon/intron splice sites. The present study showed that PBP/GOBPs in S. exigua have conserved exon/intron numbers and splice sites, as seen in the PBP/GOBPs from Agrotis moths (Picimbon & Gadenne, 2002; Abraham et al., 2005) and B. mori (Gong et al., 2009).

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Figure 5. Continued.

Expression pattern of Spodoptera exigua PBP/GOBPs Recently, several studies from lepidopteran species have shown that PBPs and GOBPs were expressed in non-antennal tissues. For example, in A. ipsilon, AipsPBPs were expressed in proboscis and labial palp (Gu et al., 2013). H. armigera PBPs and GOBPs expression were detected in legs and wings (Zhang et al., 2011; Guo et al., 2012). Similar results were also observed in non-noctuid Plutella xylostella (Sun et al., 2013) and B. mori (Zhou et al., 2009). Our previous study showed that two SexiPBPs were specifically expressed in antennae of both sexes (Xiu & Dong, 2007); however, when

doubling the cDNA concentration (one-fold increase) and PCR volume (from 25 μl to 50 μl), we found that SexiPBP1 and SexiPBP2 expression could be also detected in some non-antennal tissues; thus, our present study showed that some SexiPBP/GOBPs could be also detected in other tissues even non-olfactory tissues including proboscises, legs and heads. This is well in line with the above reported studies, suggesting functional diversities of SexiPBP/ GOBPs. Further, the three SexiPBPs had a different sex-biased expression, as indicated in the present study. In particular, in male antennae SexiPBP1 showed a much higher expression than other two SexiPBPs, similar to the PBP1 © 2014 The Royal Entomological Society

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Figure 5. Continued.

from other noctuid moths S. litura (Liu et al., 2013) and S. inferens (Jin et al., 2014), as well as the non-noctuid moth B. mori (Gong et al., 2009). As male moths can perceive the sex pheromone released by conspecific females, we suggest that the highly male-biased SexiPBP1 plays a major role in female sex pheromone reception, while the other two non-male-biased SexiPBPs possibly play minor roles. This suggestion is further supported by the results of our binding assays (see the later discussion). In S. exigua, the female begins calling on the first night after emergence and then the calling significantly increases in 1- to 3-day-old females and decreases in 4© 2014 The Royal Entomological Society

to 7-day-old females (Dong & Du, 2001). If SexiPBPs are indeed involved in female sex pheromone reception, we may expect that PBP expression would start before emergence in males. As expected, all three SexiPBPs could be detected at 2 days before emergence. This time synchronicity between male-antennal PBP synthesis and female calling has been previously reported in M. sexta, L. dispar and A. polyphemus (Györgyi et al., 1988; Vogt et al., 1989). After emergence, these three SexiPBPs show an increase in expression from 1- to 3-day-old males and then a decrease from 4- to 6-day-old males. This agedependent profile of SexiPBPs in male antennae is also well in accordance with the calling of females, further

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Table 1. Binding affinities of tested ligands to five Spodoptera exigua pheromone-binding proteins/general odorant-binding proteins PBP1

PBP2

Ligands

IC50

Ki

IC50

Acetates Z3-Hexenyl acetate Z7-14:AcP.A. Z9-14:AcP. Z11-14:AcP.A. E9-14:AcP.A. Z9,E11-14:AcP.A. Z9,E12-14:AcP. 16:AcP.A. Z9-16:AcP.A. Z11-16:AcP.

16.03 1.04 0.67 2.54 0.50 1.62 1.13 1.61 0.66 1.48

11.37 0.73 0.47 1.80 0.36 1.15 0.81 1.14 0.47 1.05

>20 >4.0 >4.0 >4.0 >4.0 4.15 3.84 >4.0 >4.0 >4.0

Aldehydes Benzaldehyde Phenylacetadehyde E2-Hexenal Z9-16:AldP.A. Z13-18:AldP.A.

>20 18.99 >40 0.73 1.31

Alcohols Phenethyl alcohol Hexanol Z3-Hexenol Linalool Geraniol Nerolidol Farnesol Z9-14:OHP. Z9,E12-14:OHP. Z11-16:OHP.

17.99 36.65 >40 >40 33.79 14.87 7.32 0.64 0.84 0.94

23.96 10.55 5.19 0.46 0.60 0.67

>20 >40 >40 39.11 >40 >40 >40 1.59 >4.0 2.79

Carboxylic acids Lauric acid Myristic acid Palmitic acid Stearic acid Oleic acid Linoleic acid

6.29 3.25 2.46 6.56 0.48 1.55

4.46 2.30 1.74 4.65 0.34 1.10

Ketones β-Ionone 2-Pentadecanone

6.13 NT

4.35 NT

13.47 0.52 0.93 12.76 25.99

>20 >40 >40 2.77 >4.0

PBP3 Ki

3.24 2.99

IC50

>20 4.03 >4.0 >4.0 >4.0 >4.0 >4.0 >4.0 >4.0 >4.0

GOBP1 Ki

3.16

IC50

>20 3.79 >4.0 >4.0 >4.0 >4.0 >4.0 >4.0 >4.0 >4.0

>20 >40 >40 >4.0 >4.0

>20 >40 >40 3.92 >4.0

2.17

>20 >40 >40 >40 >40 >40 >40 2.88 >4.0 >4.0

>20 >20 >40 >20 >20 >20 >20 4.14 >4.0 2.48

5.71 6.31 11.20 15.80 2.01 3.35

4.45 4.92 8.73 12.32 1.57 2.61

15.61 12.17 >20 >20 >20 14.00

14.12 NT

11.01 NT

12.32 NT

2.16

30.50

1.24

GOBP2 Ki

3.19

3.30

IC50

N 0.76 0.26 0.98 1.02 0.64 0.25 >4.0 1.88 >4.0 N N N 1.26 >4.0

Ki

0.50 0.17 0.65 0.67 0.42 0.17 1.24

0.83

2.10

N N N >100 >20 13.66 1.88 0.59 0.31 0.47

4.63

11.84 14.01 >20 >20 2.19 2.00

7.83 9.27

10.97

>20 >20 >20 >20 >20 5.49

9.66 NT

16.39 14.63

13.81 12.33

17.50 10.95

11.58 7.24

2.25

12.23 9.54

3.49

9.04 1.24 0.39 0.20 0.31

1.45 1.32

PBP, pheromone-binding protein; GOBP, general odorant binding protein. P. and P.A. represent sex pheromones and pheromone analogues, respectively. N means no binding. The chemicals that were not tested in binding assays are reported as ‘NT’. In the table, the concentrations of the ligands halving the fluorescence of N-phenyl-1-naphthylamine (IC50 values), which of P. and P.A. exceed 4.0 μM and other ligands exceed 20 or 40 μM, could not be obtained in the experiments. The abbreviations used are: 14:Ac, Tetradecyl acetate; 16:Ac, Hexadecyl acetate; E9-14:Ac, E9-Tetradecenyl acetate; Z11-14:Ac, Z11-Tetradecenyl acetate; Z11-16:Ac, Z11-Hexadecenyl acetate; Z11-16:OH, Z11-Hexadecenol; Z7-14:Ac, Z7-Tetradecenyl acetate; Z9,E11-14:Ac, Z9,E11-Tetradecadienyl acetate; Z9,E12-14:Ac, Z9,E12-Tetradecadienyl acetate; Z9,E12-14:OH, Z9,E12-Tetradecadienol; Z9-14:Ac, Z9-Tetradecenyl acetate; Z9-14:OH, Z9-Tetradecenol; Z9-16:Ac, Z9-Hexadecenyl acetate; Z9-16:Ald, Z9-Hexadecenal and Z13-18:Ald, Z13-Octadecenal.

suggesting SexiPBPs roles in the female sex pheromone reception. At different hours of 3-day-old males, all three SexiPBPs displayed a generally stable expression, although a small peak was observed between the late scotophase and early photophase for SexiPBP1 and SexiPBP2. This is not consistent with female calling and mating behaviour in S. exigua (Dong & Du, 2001) and other noctuid moths A. ipsilon and A. ypsilon (Gemeno & Haynes, 2000; Xiang et al., 2010); that is, females mainly called at the middle-later scotophase and did not call in

the scotophase. It seems that the expression of SexiPBPs is simply maintained during the photophase period even if the female does not call.

Binding properties of Spodoptera exigua PBP/GOBPs All five SexiPBP/GOBPs show a good affinity to the fluorescent probe 1-NPN, although these PBP/GOBPs have a low amino acid identity to each other. It is reasonable to imagine that all these proteins contain a hydrophobic binding pocket and some conserved key residues, © 2014 The Royal Entomological Society

Odorant-binding proteins in Spodoptera exigua

Figure 6. Effects of pH on the binding of Spodoptera exigua pheromone-binding proteins (PBPs)/ general odorant-binding proteins (GOBPs) to the major sex pheromone Z9,E12-14:Ac. The protein and N-phenyl-1-naphthylamine (1-NPN) were both at 2 μM. The protein solution or protein/1-NPN mixture was excited at 337 nm and emission was monitored at 400 nm. The protein/1-NPN mixture was titrated with 1.0 mM Z9,E12-14:Ac to final concentrations of 0.25–4.0 μM at pH 5.0 and 7.0.

especially aromatic amino acids. For example, the tryptophan residue (Trp37) may contribute to 1-NPN binding as a result of its aromatic nature (Bette et al., 2002). For all lepidopteran PBP/GOBPs, this tryptophan is highly conserved, and notably in B. mori this residue has been demonstrated to be very crucial for the strong binding of 1-NPN to PBP/GOBPs indicated by fluorescence resonance energy transfer (Zhou et al., 2009). It has been hypothesized that each subgroup of the PBP is tuned to a specific component(s) of the sex pheromone blend (Plettner et al., 2000; Mohl et al., 2002; Gu et al., 2013). In the present study, none of the three SexiPBPs showed an obvious specificity to any of sex pheromone components from S. exigua, as seen in the case from S. litura (Liu et al., 2012a, 2013) and other moths (Zhou et al., 2009; Guo et al., 2012); however, SexiPBP1 exhibited a much stronger affinity to all these components than the other two SexiPBPs. Further, SexiPBP1 also showed a pH-dependent conformational change mechanism, as seen in B. mori (Damberger et al., 2000). Moreover, a threshold value indicated by intrinsic fluorescence of SexiPBP1 and binding of SexiPBP1 to 1-NPN was observed at pH 6.5, consistent with the estimated pH value in the bulk sensillar lymph (Kaissling & Thorson, 1980). Taken together, these results strongly suggest that SexiPBP1 plays a major role in female sex pheromone reception. A study reported that when sex pheromone lures were added to any of the plant odorants of benzaldehyde, phenylacetaldehyde, Z3-hexenyl acetate, linalool and Z3-hexenol, S. exigua males were increasingly attracted © 2014 The Royal Entomological Society

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(Deng et al., 2004); however, the present binding studies found that none of the five SexiPBP/GOBPs exhibited an obviously strong binding to these ligands. Interestingly, three SexiPBPs, especially SexiPBP1, bound some other plant odorants with relatively high affinities. The binding of plant odorants to PBPs has already been reported from other lepidopteran species, such as A. polyphemus (Campanacci et al., 2001), Orthaga achatina (Liu et al., 2012b), S. litura (Liu et al., 2012a, 2013) and H. armigera (Guo et al., 2012). In addition, A. ipsilon PBPs were found to be expressed in female basiconic sensilla (Gu et al., 2013) that were known to involve in the recognition of plant volatiles (Vogt et al., 1991a). These results suggest the possibility that PBPs may play roles in detection of some plant odorants. By contrast, SexiGOBP2 could strongly bind sex pheromone components with comparable affinities to SexiPBP1 in the binding assays, which is consistent with the results reported in B. mori (Zhou et al., 2009), Amyelois transitella (Liu et al., 2010) and O. achatina (Liu et al., 2012b). Although early studies with A. polyphemus, A. pernyi, B. mori and M. sexta showed that GOBPs were expressed only in basiconic sensilla (Vogt et al., 1991a, 1991b), later studies using more sensitive techniques clearly showed that GOBPs were also expressed in the pheromone-sensitive long trichoid sensilla of males in M. brassicae, M. sexa, H. virescens and S. littoralis (Jacquin-Joly et al., 2000; Zhang et al., 2001; Nardi et al., 2003), therefore, we suggest that five S. exigua PBP/ GOBPs in moths have been functionally differentiated into the cross detection of plant odorants by PBPs and sex pheromones by GOBPs.

Experimental procedures Insect rearing and tissue collection. The S. exigua used in the present study were reared in the laboratory on an artificial diet (Huang et al., 2002) at 26 ± 1 °C with a photoperiod of 14 h light: 10 h dark. Pupae were sexed and were kept in separate containers. After emergence, moths were fed on 10% honey solution. For RT-PCR experiments, all tissues including antenna, proboscis, head without antenna and proboscis, thorax, abdomen (female abdomen without pheromone gland), leg and wing were dissected from 3-day-old female and male moths, respectively. Pheromone glands were collected from 3-day-old females. In daily experiments, 40 antennae were collected between the eighth and ninth hours of the scotophase from each age; in hourly experiments, 40 antennae were collected from 3-day-old male moths at different times, and immediately transferred to Eppendorf tubes immersed in liquid nitrogen. The same procedure was used for collecting larvae to extract genomic DNA. All collected these tissues were stored at −70 °C until use. Nucleic acid manipulation and cDNA synthesis. Total RNA was extracted by homogenizing antennae or other tissues of female and male moths in Trizol® reagent (Invitrogen, Carlsbad, CA,

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N.-Y. Liu et al.

USA) following the manufacturer’s instructions. The first-strand cDNA synthesis was performed with Oligo (dT)18 primer using M-MLV reverse transcriptase (TaKaRa, Dalian, Liaoning, China) at 42 °C for 60 min, and the reaction was then stopped at 70 °C for 15 min, according to the provided protocol. RACE template was synthesized using a SMART RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA) according to the manufacturer’s manual. All the templates were stored at −20 °C until use. Genomic DNA was prepared from a single fourth instar larva. The larva was disrupted by grinding in liquid nitrogen and homogenizing with 300 μl DNA extraction buffer (100 mM Tris-HCl, pH 8.0; 50 mM EDTA; 200 mM NaCl; 1% SDS). The homogenate was transferred to a clean tube, mixed with 20 μl Proteinase K (20 mg/ml) and incubated at 56 °C for 3 h. The tube was centrifuged for 10 min at 12 000 × g. The supernatant was transferred to a new tube. DNA was extracted with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) followed by an equal volume of chloroform: isoamyl alcohol (24:1), each time centrifuging for 5 min at 12 000 × g to collect the aqueous phases. Two-and-a-half volumes of chilled ethanol were added to the aqueous supernatant for DNA precipitation. The pellets were washed in 70% ethanol, briefly vacuum-dried and the DNA was re-dissolved in 50 μl TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0; 100 mg/ml RNase A). Reverse transcription PCR and quantitative real-time PCR. Based on the amino acid sequences of PBP3s and GOBP1s from H. armigera and H. assulta, orthologous PBP3 and GOBP1 fragments were identified from S. exigua transcriptome (Li et al., 2013). To obtain the full-length cDNAs of SexiPBP3 and SexiGOBP1, RT-PCR was performed using a SMART RACE cDNA Amplification Kit (Clontech) with gene-specific primers (Table S1) and Universal Primer a Mix according to the kit protocol. Expression patterns of five SexiPBP/GOBPs in female and male different tissues were assessed by RT-PCR with genespecific primers (Table S1), and specifically these primers were designed to contain two introns of genes with the product sizes of 350–400 bp. S. exigua glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (GenBank accession number: JF728815; Teng et al., 2012) was used as control to check the quality of cDNA templates, and negative control was added using sterile water as the template. The procedure was the initiation at 94 °C for 3 min, followed by 35 cycles at 94 °C for 30 s, 60 °C for 40 s, 72 °C for 40 s and final extension for 10 min at 72 °C. The same condition was also used for all five SexiPBP/GOBPs recombinant expression vector constructions. For genomic identification of SexiPBP3 and SexiGOBP1, we first ascertained the exon/intron structure of these genes based on reported lepidopteran PBP and GOBP exon/intron splice sites and then designed their primers (Table S1). Next, touchdown PCR was performed as follows: 94 °C for 3 min; 5 cycles at 94 °C for 50 s, 61 °C for 50 s and 72 °C for 2 min with a decrease of the annealing temperature by 1 °C per cycle, and followed by 35 cycles of 94 °C for 50 s, 55 °C for 50 s and 72 °C for 2 min and final extension for 10 min at 72 °C. PCR products were analysed by 1.5% agarose gels. We performed the qPCR on an ABI 7300 (Applied Biosystems, Foster City, CA, USA) with SYBR Premix Ex Taq™ (TaKaRa) using SexiGAPDH as an endogenous control. Gene-specific primers (Table S1) of all SexiPBP/GOBP genes were designed by

Beacon Designer 7.9 (PREMIER Biosoft International, CA, USA; Table S1). Each reaction well had a total volume of 20 μl, containing 10 μl of SYBR Green PCR Master Mix, 0.2 μM of each primer, 0.4 μl of ROX Reference Dye II, 2 μl of cDNA template and 6.8 μl of nuclease-free water. Each sample was run in three technical replicates (individual reaction wells) on three independent biological pools using the following PCR conditions: 1 cycle of 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Candidate gene expression was normalized to the reference gene, SexiGAPDH, using the comparative 2−ΔΔCT method (Livak & Schmittgen, 2001). In the analysis of the relative fold change, one sample which was one of the experimental samples was taken as the calibrator. For the valid ΔΔCT calculation in using the comparative 2−ΔΔCT method for relative quantification, the amplification efficiencies of the target and reference genes must be approximately equal. To confirm this, a pilot experiment was conducted to look at how ΔCT (CT,Target – CT,Actin) varies with template dilution. Briefly, four serial 10-fold dilutions of cDNA from each sample were amplified. Each dilution amplification was performed in triplicate using primers for SexiPBP/GOBPs and SexiGAPDH. The average CT was calculated for both SexiPBP/GOBPs and SexiGAPDH, and the ΔCT was determined. A plot of the log cDNA dilution vs. ΔCT was made, and the slope value of the line was calculated. The results indicated that the absolute values of the slope of all the lines were close to zero (data not shown), therefore, the efficiencies of the target and reference genes were similar, and the ΔΔCT calculation method could be used for the relative quantification. Cloning and sequencing. Purified PCR products of five SexiPBP/ GOBPs were subcloned into pEASY-T3 vector (TransGen, Beijing, China) following the manufacturer’s instructions. Positive clones were sequenced by ABI 3730 sequencer (GenScript, Nanjing, Jiangsu, China). For the construction of SexiOBPs expression vector, the plasmids containing the inserts were digested by FastDigest® restriction enzymes (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA; PBP1, PBP2, PBP3 and GOBP1: BamHI and XhoI; GOBP2: BamHI and HindIII). The expected bands were purified from agarose gels and ligated into the expression vector pET-30a (+) (Novagen, Darmstadt, Germany), previously digested with the same enzymes. Construction of the vector was confirmed by sequencing. Sequence and gene structure analysis. The putative signal peptide was predicted using the SIGNALP 4.1 program (Petersen et al., 2011). Alignments of amino acid sequences were performed using CLUSTALW2 (Larkin et al., 2007) and were visualized using JALVIEW 2.7 (Waterhouse et al., 2009). Genomic databases for B. mori (Xia et al., 2004), Danaus plexippus (Zhan et al., 2011) and Heliconius melpomene (Dasmahapatra et al., 2012) were downloaded from the Ensembl Genomes FTP Server (http://metazoa.ensembl.org/info/data/ftp/index.html). P. xylostella genomic database was downloaded from DBM-DB (http:// iae.fafu.edu.cn/DBM/; You et al., 2013). Gene structures were aligned and predicted using a GeneWise program based on the genome sequences (Birney et al., 2004). A phylogenetic tree was built with MEGA 6.06 using the Jones-Taylor-Thornton model with 1000 bootstrap replicates (Tamura et al., 2013). Expression and purification of recombinant proteins. Recombinant pET-SexiPBPs or pET-SexiGOBPs were expressed by a © 2014 The Royal Entomological Society

Odorant-binding proteins in Spodoptera exigua bacterial system in Luria-Bertani (50 μg/ml, kanamycin) with Escherichia coli BL21 (DE3) cells at 37 °C following a previously described protocol (Liu et al., 2012a, 2013). Purification of the proteins was accomplished by an affinity chromatography XK 16/20 column filled with Ni Sepharose High Performance (GE Healthcare, Little Chalfont, UK), according to the manufacturer’s protocols. The His-tag was removed by treatment with recombinant enterokinase (rEK; GenScript), and the fractions containing the target proteins were further purified and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Fluorescence binding assays. All the chemicals, including the fluorescent probe 1-NPN, were purchased from Sigma-Aldrich (purify ≥ 90%). In binding assays, ligands were prepared by dissolving in high-performance liquid chromatography purity grade methanol as 1 mM stock solutions and stored at −20 °C. To measure the affinity of 1-NPN to SexiPBP/GOBPs, a 2 μM solution of the proteins in 50 mM Tris-HCl buffer, pH 7.4, was titrated with aliquots of 1 mM 1-NPN to final concentrations of 2–20 μM. The solutions were excited at 337 nm and emission spectra were recorded at 400 nm on a Hitachi F-7000 fluorescence spectrophotometer with a 1-cm light path quartz cuvette and 10-nm slits for both excitation and emission. The tryptophan intrinsic fluorescence was measured with a 2-μM protein in 50 mM Tris-HCl buffer, pH 7.4. The excitation wavelength was 280 nm and emission spectra were recorded at 335 nm. The comparative binding of other ligands was measured using 1-NPN (2 μM) as the fluorescent reporter and 0.25–4 μM or 2–20 μM concentrations for each competitor. To evaluate the effects of pH on SexiPBP/GOBPs intrinsic fluorescence and binding of SexiPBP/GOBPs to 1-NPN or Z9,E12-14:Ac, the binding assays were performed using buffers of different pH values in 50 mM sodium acetate buffer (pH 4.0– 5.5), 50 mM ammonium acetate buffer (pH 6.0–7.0) or 50 mM Tris-HCl buffer (pH 8.0–9.0). Data analysis. To determine binding constants of 1-NPN (K1-NPN) to SexiPBP/GOBPs, the intensity values corresponding to the maximum of fluorescence emission were plotted against free ligand concentrations. Bound ligand was evaluated from the values of fluorescence intensity by assuming that the activity of proteins was 100%, with a stoichiometry of 1:1 (protein: ligand) at saturation. The curves were linearized using Scatchard plots. Dissociation constants (Ki) of each competitor were calculated from the corresponding IC50 values (the concentrations of the ligands halving the fluorescence of 1-NPN), using the following equation: Ki = [IC50]/(1 + [1-NPN]/K1-NPN), where [1-NPN] is the free concentration of 1-NPN and K1-NPN is the dissociation constant of the complex protein/1-NPN.

Acknowledgements We are very grateful to Professor Paolo Pelosi (University of Pisa) for his advice and very useful comments on the manuscript. This work was supported by a Special Fund for Agro-scientific Research in the Public Interest (201203036), funds from the National Natural Science Foundation (31071978; 30770278), and the Academic Innovative Program of Jiangsu Higher Education Institutions (CXZZ13_0304), China. © 2014 The Royal Entomological Society

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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: Figure S1. cDNA sequences and predicted amino acid sequences of S. exigua PBP3 and GOBP1. The stop codon is indicated with an asterisk. The signal peptide is underlined. The six conserved cysteines are boxed. Sites of intron 1 and 2 are marked with ‘ >