Insect Molecular Biology

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Insect Molecular Biology (2014) 23(4), 417–434

doi: 10.1111/imb.12089

The antenna-specific odorant-binding protein AlinOBP13 of the alfalfa plant bug Adelphocoris lineolatus is expressed specifically in basiconic sensilla and has high binding affinity to terpenoids

L. Sun*†, H-J. Xiao†‡, S-H. Gu†, J-J. Zhou§, Y-Y. Guo†, Z-W. Liu* and Y-J. Zhang† *Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing, China; †State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China; ‡Institute of Entomology, Jiangxi Agricultural University, Nanchang, China; §Department of Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, UK Abstract Odorant-binding proteins (OBPs) are crucial in the olfactory pathway of insects. In the present study, the antenna-enriched OBP AlinOBP13 was investigated because of its potential contribution to the peripheral olfactory perception in the alfalfa plant bug Adelphocoris lineolatus. The results of quantitative reverse transcriptase-PCR showed that the transcript level of AlinOBP13 was higher in the adult stage than in the nymph stages. The transcript levels of AlinOBP13 in the male and female antennae significantly increased after 4 and 8 h of starvation, respectively. Fine ultrastructures of different types of chemosensilla in both female and male antennae were investigated using transmission electron microscopy and immunocytochemical labelling. The results

First published online 28 February 2014. Correspondence: Yong-Jun Zhang, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China. Tel.: +86 10 62815929; fax: +86 10 62816631; e-mail: [email protected] Ze-Wen Liu, Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China. Tel.: +86 25 84399051; fax: +86 25 84399051; e-mail: [email protected]

© 2014 The Royal Entomological Society

revealed that the anti-AlinOBP13 antiserum strongly and specifically labelled short basiconic sensilla; this antiserum was restricted to the inner lumen and the cavities below the sensillum base of the sensilla. By contrast, multiporous sensilla trichodea, medium long sensilla basiconica, and aporous sensilla chaetica were not labelled. The present study is the first to report an OBP showing specific expression in the short basiconic sensilla of a member of the Hemipteran species. The results of a fluorescence displacement binding assay indicated that recombinant AlinOBP13 showed a more specific binding preference to terpenoids than to sex pheromones and other classes of chemicals. This binding ability was dramatically affected by pH; higher binding affinities were displayed at pH 10.0 than at pH 7.4 and 5.0. In addition, the results of dose-dependent electroantennogram recordings from the antennae showed that both female and male adult bugs responded to the terpenoids tested, suggesting an apparent physiological relevance of AlinOBP13 in A. lineolatus chemoreception. The results of this study suggest that AlinOBP13 functions as a specific carrier of terpenoids and provide insights into the mechanism of A. lineolatus in response to green volatiles. Keywords: Adelphocoris lineolatus, odorant binding protein, fluorescence competitive binding assays, pH-dependent ligand binding, immunocytochemistry, host plant volatile perception, electroantennogram recording.

Introduction Semiochemicals in the environment, such as host plant volatiles and sex pheromones, are crucial for the survival and reproduction of insects (Fatouros et al., 2012; 417

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Matsuura, 2012). Sex pheromone blends are usually produced by females in a certain species-specific ratio to attract conspecific males and achieve successful mating (Karlson & Lüscher, 1959; Aldrich, 1988). Host plant volatiles, especially terpenoids, mediate insect activities, such as feeding, oviposition, aggregation, dispersion and defence (Arimura et al., 2009; Xiao et al., 2012). Through the course of their long evolutionary period, insects have developed a sophisticated olfactory system to perceive and discriminate these vital chemical cues. Insect antennae are equipped with various chemosensilla where specialized sensory neurons are innervated (Schneider, 1964). Different types of olfactory neurons are grouped in the sensilla in morphologically and physiologically welldefined units (Keil, 1999). In insects, each of these olfactory neurons within a sensillum is tuned to one biologically significant compound (Meng et al., 1989). Hydrophobic odorants must pass through the aqueous sensillum lymph to activate olfactory receptor neurons. Insect soluble odorant-binding proteins (OBPs), which are extremely abundant in the sensillum lymph (up to 10 mM), are believed to be involved in this process, by solubilizing and transporting lipophilic odorants across the sensillum lymph to odorant receptors in the dendritic membranes of olfactory receptor neurons (Kaissling et al., 1991; Van den Berg & Ziegelberger, 1991; Pelosi et al., 2006; Zhou, 2010; Leal, 2012; Kaissling, 2013). It has been proposed that insect OBPs are synthesized by the endoplasmic reticulum in the auxiliary cells (mainly trichogen and tormogen cells) of chemosensilla and are secreted into the sensillum lymph (Vogt & Riddiford, 1981; Klein, 1987). Numerous studies have investigated the specific expression of OBPs in different types of antennal sensilla and the association of this expression with the pheromone-sensitive sensilla trichodea and general odorant-sensitive sensilla basiconica (Steinbrecht et al., 1995; Shanbhag et al., 2001; Zhang et al., 2001; Gu et al., 2013). In addition, efforts have been made to characterize the ultrastructures of different antennal sensilla and the co-expression of distinct OBPs in the same sensilla (Shanbhag et al., 2001; Jin et al., 2006; Li et al., 2011; Deng et al., 2012; Sun et al., 2012a) and to measure the binding abilities of OBPs to diverse compounds using different biochemical methods in vitro (Vogt & Riddiford, 1981; Ban et al., 2002; Hooper et al., 2009; Zhou et al., 2009; Hua et al., 2012; Sun et al., 2013a). The mechanism of ligand binding and release is another interesting aspect of OBP research. Various studies suggested that Lepidopteran pheromone-binding proteins (PBPs), a subgroup of OBPs, undergo pH-dependent conformational changes associated with a dramatic decrease in binding affinity at acid pH, which facilitates the release of odorants to receptors (Kowcun et al., 2001;

Zubkov et al., 2005). A recent study on the binding ability of two minus-C OBPs of Helicoverpa armigera has revealed a contrary pH-dependent binding behaviour (Li et al., 2013). By contrast, structural studies on general OBPs, another subgroup of OBPs, found no conformational changes in Bombyx mori BmorGOBP2 complexed with sex pheromones and their analogues (Zhou et al., 2009). The alfalfa plant bug Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae) is a destructive pest that affects many crops (Lu & Wu, 2008). With the adoption of broadspectrum transgenic Bacillus thuringiensis cotton, this piercing-sucking pest and other mirids have evolved into key pests of cotton crops in Northern China (Lu et al., 2010). Frequent overlapping generations (Wu et al., 2002), fine-flight capacity (Lu et al., 2009), and various host plants (Lu & Wu, 2008) have worsened this situation. Plant volatiles, especially terpenoids, are crucial in A. lineolatus host plant or oviposition site location. Thus, a better understanding of the molecular and cellular mechanisms of these odorants perception will largely contribute to the integrated management of A. lineolatus. A previous study has identified and investigated the tissue distribution of 14 OBP genes in A. lineolatus based on antennal cDNA library construction; of these genes, AlinOBP1-6, AlinOBP8, AlinOBP12, and AlinOBP13 are highly expressed in the adult antennae (Gu et al., 2011a). Other studies found, using competitive fluorescence binding assays, that four of these antennal highly expressed OBPs (i.e. AlinOBP1, AlinOBP2, AlinOBP3 and AlinOBP5), and AlinOBP10 which has also been shown to be antenna-specific, can interact with various semiochemicals (Gu et al., 2010, 2011b, c; Sun et al., 2013b; Wang et al., 2013). Given that different OBPs may cooperatively interact with the same odorant (Sun et al., 2012b) or are preferentially responsible for distinct stimuli perception (Große-Wilde et al., 2006), the physiological functions, particularly the binding profiles of other OBPs of A. lineolatus should be well studied. The present study determined the potential contribution of another antenna-enriched odorant binding protein, AlinOBP13, to the peripheral molecular perception of host plant volatiles in A. lineolatus. The ultrastructures of different types of antennal sensilla and the specific sensillum distributions of AlinOBP13 protein were examined at the cellular level using transmission electron microscopy (TEM) and immunocytochemical labelling. The binding specificity of AlinOBP13 to a wide range of chemicals was investigated using fluorescence competitive binding assays. The effect of pH on the binding affinity of AlinOBP13 was also analysed. The electrophysiological responses of the antennae to putative ligands of AlinOBP13 were confirmed using an electroantennogram (EAG) recording assay. © 2014 The Royal Entomological Society, 23, 417–434

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Results

Ultrastructures of different antennal sensilla in A. lineolatus

Expression of AlinOBP13 transcription during developmental stages and the effect of starvation

Using scanning electron microscopy, Gu et al. (2012) found at least seven types of sensilla on A. lineolatus antennae: two types of sensilla basiconica: medium-long sensilla basiconica and short sensilla basiconica; two types of sensilla trichodea: long straight sensilla trichodea and long curved sensilla trichodea; two types of sensilla chaetica: long straight sensilla chaetica and long curved sensilla chaetica; and Böhm bristles. In the present study, the ultrastructures of these types of chemosensilla on both female and male antennae were further examined in detail using TEM. No significant difference in the ultrastructure of each type of sensilla was found between sexes. Two types of sensilla basiconica exhibited significantly different ultrastructures. Both cross-section and longitudinal sections showed that medium-long sensilla basiconica was a single-walled sensillum and that the cuticular wall was extremely thin with numerous pores sculptured on. Numerous branched dendrites with clear microtubules were observed suspended at the sensillum lymph (Fig. 2A–D). By contrast, cross-sections of short sensilla basiconica revealed a double-walled organization (Fig. 3A–F). The outer cuticular wall and the inner cuticular wall formed the outer and inner sensillum lumina,

Quantitative reverse transcriptase (qRT)-PCR was used to investigate the effect of different developmental stages and starvation on the expression of AlinOBP13. The results showed that the transcript level of AlinOBP13 decreased from the first to the second instar nymphal stages, remained relatively low from the second to the fifth instar nymphal stages, and then increased at the newly emerged adult stage. The relative expression level at the adult stage was approximately two fold higher than those at the different nymphal stages (Fig. 1A). Furthermore, a significant correlation was found between the increase in AlinOBP13 transcript level and starvation in both sexes. The relative expression level of AlinOBP13 significantly increased in the antennae of female adults starved for 8 h (Fig. 1C). The transcript level of AlinOBP13 in male antennae started to increase 4 h earlier than that in female antennae and remained higher during the next 4 h after starvation (Fig. 1B). At 12 h starvation, the transcript expression of AlinOBP13 in male and female antennae similarly reached the levels observed before starvation.

Figure 1. Relative expression levels of AlinOBP13 transcripts in different developmental stages (A) and different starvation intervals of both sexes (B and C). Fold changes in developmental stages and different starvation intervals are relative to the transcript levels of the first instar and 0 h starvation in female or male antennae, respectively. The standard error is represented by the error bar, and the different letters above each bar denote significant differences (P < 0.05). *P < 0.05, **P < 0.01.

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Figure 2. Ultrastructures of medium-long sensilla basiconica in Adelphocoris lineolatus adults of males. No significant difference in the ultrastructures of this sensillum was observed between males and females. (A–B) Longitudinal section indicated numerous pores (p) on the thin cuticular wall (cw) and several branched dendrites (d) located in the sensillum lymph. (C–D) Cross sections on the top and base of the sensillum shaft showed numerous dendrites and clear microtubules (mt) on each dendrite.

respectively. The inner sensillum cavity was surrounded by the inner cuticular wall, and five unbranched sensory dendrites with microtubules were located in the inner sensillum lymph (Fig. 3F). Various spoke channels lead the inner sensillum-lymph cavity to the groove channels; consequently, the external environmental substances gain access to the inner sensillum lumen (Fig. 3B–E). Two types of sensilla trichodea showed a similar inner structure of a single-walled sensillum with a thick cuticular

wall; in addition, various clear cuticular pores were observed in cross-sections and longitudinal sections (Fig. 4A–D). Different subtypes of these sensilla can be distinguished based on the distinct number of sensory dendrites in the sensillum lymph. Two distinct sensilla chaetica were found at the antennae of A. lineolatus (Fig. 4E–H and Fig. 4I–L). Crosssectional analysis showed that long curved sensilla chaetica have two sensillum chambers separated by a

Figure 3. Ultrastructures of short sensilla basiconica in male Adelphocoris lineolatus adults. No significant difference in the ultrastructures of this sensillum was observed between males and females; (A–B) Longitudinal sections showed a double-walled organization connected by several spoke channels (sc). (C–E) Cross sections at the different degrees of sensillum shaft displayed the outer cuticular wall (ow) and the inner cuticular wall (iw), which formed the outer and inner sensillum lumina, respectively, and the inner sensillum cavity (isl) was surrounded by the outer cuticular wall (osl) and led by the sc to grooved channels (gc). (F) Cross sections at the base of short sensilla basiconica unambiguously revealed five dendrites (d) surrounded by the dendrite sheath (ds).

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Figure 4. Ultrastructures of sensilla trichodea and sensilla chaetica in male Adelphocoris lineolatus adults. No significant difference in the ultrastructures of these sensilla was observed between male and females. (A) Longitudinal section of the sensilla trichodea hair. (B–D) Cross sections of the sensilla trichodea showed a thick cuticular wall penetrated by various pores (p) and one, two or more dendrites in the sensillum with clear microtubules (mt). (E) Longitudinal section on the long curved sensilla chaetica shaft showed socket(s) at the base of the sensillum and a clear dendrite (d) at the sensillum cavity. (F–H) Cross sections of long curved sensilla chaetica showed that two sensillum chambers (isl and osl) were separated by dendrite sheath (ds) and that no pore was present on the cuticular wall (cw). (I) Longitudinal section of long straight sensilla chaetica showed socket (s) at the base of the sensillum. (J–L) Cross sections on the different degree of the sensillum shaft indicated a clear socket (s), thick cuticular wall (cw), and no dendrite was observed on the sensillum lymph (isl).

dendrite sheath; no pore was observed on the cuticular wall, and one dendrite was housed in the cavity of the sensillum (Fig. 4E–H); however, long straight sensilla chaetica showed a single-walled sensillum with a very thick cuticular wall, and no pores but a conspicuous groove was found; meanwhile, cross-section of the bottom of the sensilla revealed no sensory dendrite (Fig. 4I–L). The ultrastructure of Böhm bristles was not characterized in the present study.

Specific localization of AlinOBP13 in antennal sensilla of A. lineolatus We prepared a polyclonal antiserum against recombinant AlinOBP13 and investigated the immunolocalization in distinct antennal sensilla of A. lineolatus based on the aforementioned descriptions of sensillum ultrastructures. The specificity of the antibody was verified by Western blot analysis, and the results showed that anti-AlinOBP13 reacted with the purified recombinant AlinOBP13 (Fig. 5A). Meanwhile AlinOBP13 was showed to be expressed in both male and female antennae (Fig. 5B). Immunocytochemical labelling results revealed that AlinOBP13 had an unusual specific distribution among the different A. lineolatus adult chemosensilla. The short basiconic sensilla were strongly and specifically stained (Fig. 6A–F). By contrast, no gold granules were observed in the sensilla © 2014 The Royal Entomological Society, 23, 417–434

trichodea and sensilla chaetica, and very weak or no labelling was observed at the medium-long sensilla basiconica (Fig. 6G–K). Cross-sections at different positions of the hair sensilla of short sensilla basiconica were analysed. As shown in Fig. 6A–C, the inner sensillum lumen was very strongly labelled, but the outer sensillum lumen, sensory dendrites and cuticular wall were not labelled. Longitudinal sections showed that both the sensillum lymph in the sensillum peg lumen and the cavities below the peg base were strongly labelled (Fig. 6D,E). In addition, the auxiliary cells (probably trichogen cells) of short basiconic sensilla were also clearly labelled (Fig. 6F).

Binding specificity of recombinant AlinOBP13 The specific expression of AlinOBP13 in short basiconic sensilla combined with a reported olfactory function of this sensillum in Hemipteran species (Diehl et al., 2003) may suggest that AlinOBP13 is involved in olfactory recognition processes, such as the binding of semiochemicals, as demonstrated in other OBPs (Gu et al., 2011b; Wang et al., 2013). We used the purified recombinant protein to illustrate the binding specificity of AlinOBP13 using N-phenyl-1-naphthylamine (1-NPN) as the fluorescence probe in the competitive binding assay, which displayed a strong blue shift followed by a six-fold increase in fluorescence intensity when bound to AlinOBP13 (Fig. S1). The

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Figure 5. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of expressed recombinant AlinOBP13 and antennal total proteins. (A) M. Molecular weight marker; 1. Non-induced PET/AlinOBP13; 2. Induced PET/AlinOBP13; 3. Supernatant of PET/AlinOBP13; 4. Pellet of PET/AlinOBP13. 5. Purified AlinOBP13 with His-tag. 6. Purified AlinOBP13 cleaved His-tag by rEK. 7. Western Blot analysis of the purified AlinOBP13 using polyclonal rabbit antiserum. (B) Western blot analysis of AlinOBP13 expression in total protein extracts of male and female antennae of Adelphocoris lineolatus. PET, PET-30a(+) expression vector.

dissociation constant of the AlinOBP13/1-NPN complex was 8.99 μM, as calculated by Scatchard plot (Fig. S2). The recombinant AlinOBP13 showed a very specific binding profile. The tested chemicals, including aliphatic alcohols, aldehydes, ketones, aromatic compounds and most aliphatic esters, could not compete with 1-NPN for feasible binding to AlinOBP13 (dissociation constant Ki > 50 μM, Fig. 7A–C). In addition, five sex pheromone analogues and some aromatic compounds could not displace 1-NPN from the AlinOBP13/1-NPN complex even if their concentration reached up to 50 μM (Fig. 7D, F). By contrast, AlinOBP13 exhibited a greater binding preference to several terpenoids, with Ki values ranging from 12 μM to 20 μM (Fig. 7E, and Fig. 8, Table 1).

Based on the binding results of Table 1 and Fig. 7, all tested terpenoids were selected to investigate the effect of pH on the binding ability of AlinOBP13. The binding curves and the Scatchard plots in Fig. S3 showed that different pH levels (5.0, 7.4 and 10.0) significantly affected the binding between 1-NPN and the recombinant AlinOBP13. AlinOBP13 showed higher binding preference to 1-NPN at an alkaline pH than at a neutral and acidic pH. Further competitive binding assays of the selected terpenoids showed lower binding affinities at acidic pH and considerably higher binding abilities at alkaline pH (Fig. 9). The binding affinities of some terpenoids decreased significantly when pH was decreased from 7.4 to 5.0; however, the binding of the recombinant AlinOBP13 to α-phellandrene, β-pinene, β-ionone, trans-β-farnesene, and trans, trans-farnesol was not significantly different between pH 5.0 and 7.4. Interestingly, all terpenoids exhibited dramatically better binding to AlinOBP13 at alkaline pH than at neutral and acidic pH; with 1/Ki at least twoto threefold higher at pH 10.0 than at pH 7.4 and 5.0, respectively (Fig. 9). Distinct buffers (sodium acetate for pH 5.0 and Tris-HCl for pH 10.0 and 7.4) were used to provide the different pH conditions; the binding differences may therefore be attributed to the sodium ion. A binding assay at pH 7.4 with different sodium ion concentrations (0, 30 and 60 mM) was conducted for β-caryophyllene and trans-β-farnesene. No obvious differences in the effects of the different sodium ion concentrations were observed (Fig. S4). Another purified OBP of A. lineolatus, AlinOBP11, and a sex pheromone binding protein of Helicoverpa armigera, HarmPBP1, were used as control proteins to verify the effect of pH on AlinOBP13 binding. AlinOBP11 exhibited a similar higher binding ability to its ligand at pH 10.0 than pH 7.4, whereas HarmPBP1 showed an approximately equivalent binding profile between pH 10.0 and 7.4 (Fig. S5).

Electroantennogram recordings of putative ligands of AlinOBP13 We measured the electrophysiological responses of A. lineolatus antennae to these terpenoids and performed EAG recording experiments to examine whether or not these terpenoids exhibit biological activities and to identify the possible involvement of AlinOBP13 in the perception of these terpenoids. The results clearly indicated that all the tested terpenoids could elicit EAG responses at varying degrees to both male and female antennae, in which nerolidol elicited the highest EAG response among all ligands at the 1−1 and 10−1 levels (Fig. 10). Except for © 2014 The Royal Entomological Society, 23, 417–434

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Figure 6. Immunocytochemical localization of AlinOBP13 among different antennal sensilla from male Adelphocoris lineolatus adults. The immunolocalization in the antennal sensilla of female Adelphocoris lineolatus was similar to that in the antennal sensilla of male A. lineolatus. The inner sensillum lymph, the auxiliary cells, and the cavity below the peg base of the short sensilla basiconica were strongly labelled. By contrast, no clear labelling was observed at the out sensillum lymph sensory dendrites or the cuticular wall of the short sensilla basiconica (cross sections A, B, C and longitudinal sections D, E, and F). Multiporous sensilla trichodea medium-long sensilla basiconica and aporous sensilla chaetica were never labelled (cross sections G, H, I, J, and K). A few grains found over the cuticular wall and the dendrites represented nonspecific background. The dilution of the primary antibody of AlinOBP13 was 1:2500, and the secondary antibody was anti-rabbit IgG conjugated with 10 nm colloidal gold granules at 1:20 dilution.

β-caryophyllene, all other terpenoids exhibited significantly sexual dimorphism at the 10−1 level, with the male antennae being more responsive to trans-β-farnesene and the female antennae being more sensitive to the rest of the terpenoids (Fig. 10). Three-dimensional model structure Considering the low sequence similarity of AinOBP13 to any insect OBPs with known structures, the threedimensional (3D) model was predicted by the platform of iterative threading assembly refinement (I-TASSER) server. The verification score of the final model using PROFILES-3D was 40.85, which is considerably higher than the expected low verification score of 24.19, and 96.2% of residues were located at the rational region based on the dihedral angle distributions of amino acid in the backbone assessed by Ramachandran profile, implying that the overall quality of the predicted structure was reliable. The predicted 3D model of AlinOBP13 was composed of six α-helices located between residues Ser3-Glu23 (α1), Gly28-Gly35 (α2), Thr39-Lys52 (α3), Met65-His75 (α4), Ala81-Lys99 (α5), and Thr103-Lys117 (α6), in which three pairs of disulphide bridges formed by Cys20-Cys48, Cys44-Cys104, and Cys93-Cys113 connected α1-α3, © 2014 The Royal Entomological Society, 23, 417–434

α3-α6, and α5-α6, respectively (Fig. S6). The interlocked disulphide bridges maintained the stability of the tertiary structure. The established structure of AlinOBP13 displayed a large binding protect, in which many hydrophobic residues were located in Vla89, Vla90, Leu68, Phe85, Leu71, Ser107, Ile72, Ser49, Leu50 and Pro119. Meanwhile, several hydrophilic residues, including Arg6, His75, Lys86, Lys117, and Asp9, also appeared in the binding pocket (Fig. S6). Discussion Odorant-binding proteins are the first elements to interact with environmental odorants and thus have crucial functions in odorant recognition and transportation (Pelosi et al., 2006). Previous work has reported that the transcript of AlinOBP13 was mainly restricted to female and male antennae (Gu et al., 2011a), implying a potential role of this protein involved in olfactory chemoreception. The results of qRT-PCR indicated that AlinOBP13 showed different levels of transcription during the different developmental stages of the plant bug. The transcript level of AlinOBP13 was higher in the adult stage than in other developmental stages. These results were consistent with those found in other A. lineolatus OBPs, i.e. AlinOBP1

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Figure 7. Competitive binding curves of AlinOBP13 to odorants with different functional groups (A, B, C, D, E and F represent aliphatic alcohols, aldehydes, ketones, esters, terpenoids, and other compounds, including undecane indole and aromatic compounds). A mixture of the recombinant protein and N-phenyl-1-naphthylamine (1-NPN) in 50 mM Tris-Hcl buffer (pH 7.4) both at the concentration of 2 μM was titrated with 1 mM solutions of each competing ligand to the final concentration range of 2 μM to 50 μM. Fluorescence intensities are reported as percent of the values in the absence of competitor. Data are represented as means of three independent experiments.

and AlinOBP10 showed higher expression levels in the adult stage than in other developmental stages (Gu et al., 2011b; Sun et al., 2013b). Female adults of A. lineolatus lay their eggs on their host plants, therefore, nymphal bugs do not require moving and searching for feeding; however, adults sometimes need to migrate for host plants (Lu & Wu, 2008; Lu et al., 2009). Indeed OBP expression levels are reported to be regulated by feeding status (Biessmann et al., 2005; Badonnel et al., 2007; Liu et al.,

2010). In Glossina morsitans morsitans, there was a clear correlation between OBP gene transcription levels and feeding status, and the transcriptions of some OBP genes in female antennae were upregulated by >48 h of starvation after the first blood meal (Liu et al., 2010). The present results revealed an upregulated transcription of AlinOBP13 by starvation, but the increase in the transcript levels of OBP genes from A. lineolatus occurred considerably earlier than those of OBP genes from tsetse fly, © 2014 The Royal Entomological Society, 23, 417–434

Immunolocalization and binding of AlinOBP13

Figure 8. Comparison of binding ability [indicated by 1/Ki (dissociation constant) values] of AlinOBP13 with 14 ligands (13 terpenoids and other three compounds whose IC50 < 50). Data are represented as means of three independent experiments, and the standard error is represented by the error bar.

with 8 h in female antennae and 4 h in male antennae after starvation. These results indicated that AlinOBP13 expression was closely related to the physiological desire for food; however, the transcription of AlinOBP13 decreased to the level observed before starvation. This decrease in transcript level may be attributable to the decrease in insect physiological condition after 12 h of starvation. Various types of sensilla equipped on the antennae enable insects to detect and discriminate distinct odorants (Schneider, 1964). Chemosensilla in Hemipteran species have been characterized (Chinta et al., 1997; Lu et al., 2007; Romani & Stacconi, 2009). Recently, at least seven different sensilla organs have been reported in both sexes of A. lineolatus (Gu et al., 2012), but the ultrastructures of these sensilla have not been reported yet. The present TEM results revealed that different antennal sensilla had significantly different ultrastructures. The trichoid sensilla sensilla trichodea had fewer dendrites in the sensillum lymph compared with numerous branched dendrites observed at the sensillum lymph of medium-long sensilla basiconica; however, both of these two types of hair sensilla displayed abundant pores on the single cuticular wall, indicating a potential olfactory function. In Lepidopteran species, long trichoid sensilla are considered sensitive to sex pheromones, whereas basiconic sensilla are generalized to green odorants (Schneider, 1964; Steinbrecht, 1997). Another OBP in A. lineolatus, AlinOBP1, was found located on long trichoid sensilla and medium-long sensilla basiconica and displayed high binding affinities to both sex pheromone analogues and many important host plant volatiles (Gu et al., 2011b). It is possible that A. lineolatus sensilla trichodea could poten© 2014 The Royal Entomological Society, 23, 417–434

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tially be equipped for the detection of sex pheromones, and medium-long sensilla basiconica may be responsible for the perception of general odorants. Unlike sensilla trichodea and medium-long sensilla basiconica, short sensilla basiconica were double-walled and possessed no obvious pores on the cuticular wall; however, the inner sensillum-lymph cavity may connect to the external environment through the groove channels and various spoke channels. This ultrastructure was similar to the short peg of Lygus lineolaris and the grooved-peg in Triatoma infestans (Chinta et al., 1997; Diehl et al., 2003). An olfactory function has been demonstrated by the electrophysiological investigation in T. infestans, suggesting that this sensillum can respond to aliphatic amines (Chinta et al., 1997; Diehl et al., 2003). The results of immunocytochemical labelling with anti-AlinOBP13 antiserum showed that AlinOBP13 was specifically located in the inner lumen and the cavities below the hair base of short sensilla basiconica. To the best of our knowledge, the present study is the first to reveal the specific localization of an OBP from a member of the Hemipteran species in short sensilla basiconica. High expression of olfactory protein and the presence of various spoke channels combined with the reported olfactory function in T. infestans imply that the short sensilla basiconica in A. lineolatus have a potential olfactory function and that AlinOBP13 has a putative function in olfactory perception such as solubilizing odorants, transporting hydrophobic molecules to olfactory receptors and increasing the sensitivity and specificity of the olfactory system to odours. The above findings are further supported by binding experiments with AlinOBP13, in which all the tested terpenes can interact with AlinOBP13, with higher binding affinities compared with other semiochemicals. The host plants of A. lineolatus, such as cotton and alfalfa, can produce most of these terpenoids naturally or upon herbivore attack (Loughrin et al., 1994; Röse & Tumlinson, 2004). In addition, some terpenoids usually function as cues for some herbivores and their natural enemies to locate their hosts or prey (Büchel et al., 2011). In the present study, the biological activities of these terpenoids from the antennae of male and female A. lineolatus, as demonstrated by EAG recordings, supported this binding behaviour and suggested the significant physiological relevance of AlinOBP13 in response to terpene volatiles. Odorant-binding proteins are known to solubilize odorants, transport hydrophobic molecules to odorant receptors, and increase the sensitivity of the olfactory system (Pelosi et al., 2006; Leal, 2012); however, whether or not OBPs can discriminate different groups of chemicals and subsequently contribute to the selectivity of insect olfactory coding remains unclear (Leal, 2012). The binding properties of OBPs in A. lineolatus, such

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Ligand Aliphatic alcohols Hexanola,b 2-Hexanolc Pentanold cis-3-Hexen-1-ole Aliphatic aldehydes Valeraldehydef Heptanald,f Octanalf trans-2-Hexenala,e,f Hexanalg Nonanalg Aliphatic ketones 2-Hexanonef 2-Octanonec 3-Hexanonef 2-Heptanonef 6-Methyl-5-hepten-2-onef Aliphatic esters Amyl acetatef Nonyl acetatef Butyl butyrateh trans-2-hexenyl butyrateh Hexyl butyrateh Hexyl hexanoatei Ethyl butyrateh cis-3-hexenyl acetatea,b,e Terpenoids Limonenea α-Phellandrenek β-Pinenea,e,f (+)-α-Pinenea,e,f β-Iononej Myrcenea,e Nerolidolj β-Caryophyllene a α-Humulenea trans-β-Farnesenea trans,trans-Farnesolk Others Undecanef Indolea,b,e Benzaldehydef 3,4-Dimethyl-benzaldehydek Acetophenonef Methyl salicylatee,f

CAS Number

IC50 (μM)

Ki (μM)

111-27-3 626-93-7 71-41-0 928-96-1

>50 >50 >50 >50

>50 >50 >50 >50

110-62-3 111-71-7 124-13-0 6278-26-3 66-25-1 124-19-6

>50 >50 >50 >50 >50 >50

>50 >50 >50 >50 >50 >50

591-78-6 111-13-7 589-38-8 110-43-0 110-93-0

>50 >50 >50 >50 >50

>50 >50 >50 >50 >50

628–637-7 1143-13-5 109-21-7 53398-83-7 2639-63-6 6378-65-0 105-54-4 3681-71-8

>50 >50 >50 47.8 ± 2.16 >50 >50 >50 46.56 ± 0.25

>50 >50 >50 39.86 ± 1.80 >50 >50 >50 38.71 ± 0.24

5989-27-5 99-83-2 18172-67-3 7785-70-8 79-77-6 123-35-3 7212-44-4 87-44-5 6753-98-6 18794-84-8 106-28-5

21.82 ± 1.32 24.32 ± 1.60 19.10 ± 1.07 21.83 ± 1.26 23.46 ± 0.64 19.61 ± 1.18 17.47 ± 0.76 16.65 ± 0.29 16.71 ± 0.64 15.34 ± 0.28 15.61 ± 0.40

18.21 ± 1.34 20.29 ± 1.38 15.87 ± 0.88 18.20 ± 1.08 19.50 ± 0.54 16.33 ± 1.00 14.52 ± 0.62 13.84 ± 0.24 13.88 ± 0.52 12.77 ± 0.24 12.99 ± 0.45

1120-21-4 120-72-9 100-52-7 5973-71-7 98-86-2 119-36-8

>50 >50 >50 >50 >50 47.96 ± 0.54

>50 >50 >50 >50 >50 39.91 ± 0.49

Table 1. Binding data of recombinant AlinOBP13 to compounds with different functional groups. IC50 and Ki > 50 indicate that IC50 or Ki cannot be accurately calculated at the ligand concentration range tested in the assay

IC50, Ligand concentration displacing 50% of the fluorescence intensity of the AlinOBP13/1-NPN complex; Ki, dissociation constant. a, b, c,d, e, f, g, h, i, j, k represent references: Loughrin et al. (1994); Williams et al. (2010); Bengtsson et al. (2001); Van Straten & Maarse (1983); Blackmer et al. (2004); Yu et al. (2007); Chinta et al. (1994); Aldrich (1988); Millar (2005); Utama et al. (2002); Pan et al. (2010), respectively.

as AlinOBP1, AlinOBP2, AlinOBP3, AlinOBP5 and AlinOBP10, have been previously dissected (Gu et al., 2010, 2011b, c; Wang et al., 2013; Sun et al., 2013b). AlinOBP1 can bind to various chemicals with different functional groups, such as plant volatiles alcohols, aldehydes, ketones and terpenes, as well as the putative bug sex pheromones (i.e. esters). Meanwhile, AlinOBP2, AlinOBP3, AlinOBP5 and AlinOBP10 can also interact with a series of semiochemicals with different binding affinities. Unlike other OBPs characterized in A. lineolatus, AlinOBP13 was found to have a specific

binding preference to terpenoids over the other tested compounds. Further comparison of the immunolocalization and binding profiles between AlinOBP1 and AlinOBP13 indicated a possible correlation between OBP expression and its binding preference. AlinOBP1 was immunolocated in both sensilla trichodea and mediumlong sensilla basiconica, and can bind to both Miridae sex pheromones and various plant volatiles. By contrast, AlinOBP13 was only heavily immunolocated at short sensilla basiconica, and its binding profile was restricted to terpenoids. Binding specificity has always been attri© 2014 The Royal Entomological Society, 23, 417–434

Immunolocalization and binding of AlinOBP13

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Figure 9. Comparison of binding affinities (indicated by 1/Ki value) of AlinOBP13 with terpenoids at three pH values. The standard error is represented by the error bar, and the different letters above each bar denote significant differences (P < 0.05).

buted to ligand structure and OBP spatial structure, as well as their specific interactions (Pelosi et al., 2006). Recently, the possible mechanism of AlinOBP5 binding to cis-nerolidol has been revealed by molecular simulation and site-directed mutagenesis. The results of these methods have suggested that two key amino acid residues, Lys74 and Pro121, are involved in the recognition of cis-nerolidol. That is, Lys74 may form a hydrogen bond with the hydroxyl group of cis-nerolidol, and Pro121 may contribute to valid C-terminus folds inside the central cavity. Qiao et al. (2009) proposed that some residues, such as Ile43, Leu48, Leu64, Val104, and Leu108, on opposite α-helical domains may be a reasonable explanation for the specific binding behaviour of aphid OBP3 to trans-β-farnesene. Several hydrophilic residues (such as Arg6, His75, Lys86, Lys117 and Asp9) and hydrophobic residues (such as Ile72, Leu68, Leu71, Leu50, Leu110, Vla89 and Vla90) were predicted to be the putative binding sites of AlinOBP13. These residues may interact with functional groups and the branched chains of terpenoids, there by accounting for the specific binding preference of AlinOBP13 to terpenoids. The pH-dependent mechanism of ligand binding and release involving C-terminal conformational change in Lepidopteran PBPs has been demonstrated by biochemical, biophysical, structural and kinetics studies (Horst et al., 2001). The effect of pH on the binding affinities of a Hemipteran OBP, AlinOBP13, was evaluated in the present study. The results showed that acidic pH (pH 5.0) did not always affect the terpenoid binding abilities. By contrast, considerably higher binding affinities were obtained at pH 10.0. This result was not attributed to the different sodium ions used in the binding buffers and supported by the binding results of two other OBPs, namely, AlinOBP11 and HarmPBP1. In the silkworm moth B. mori, the C-terminal region of PBP forms an α-helix that competes with the pheromone by inserting into the binding © 2014 The Royal Entomological Society, 23, 417–434

pocket at low pH; however, this C-terminal region becomes unstructured when the pheromone binds to PBP at a high pH revealed by the bombykol-BmorPBP structure (Sandler et al., 2000; Lautenschlager et al., 2005, 2007). In Amyelois transitella, the C-terminal region of PBP1 forms an α-helix that competes with pheromone binding at low pH; deletion of the entire C-terminal helix (residues 129 to 142) increases the pheromone-binding affinity by > 100-fold (Xu et al., 2011). To date, no crystal structure of a Hemipteran OBP has been analysed, and whether or not a conformational change in the C-terminal region at different pH conditions is responsible for the significantly increasing binding affinities of AlinOBP13 to terpenoids at alkaline pH remains unknown. The mechanism of ligand binding and release is based on the interaction between protein and its ligands, in which key amino acid residues located at the binding cavity may have crucial functions (Pelosi et al., 2006). Histidine protonation switch is a key factor in the mechanism of pheromone binding and release of AtraPBP1, wherein two salt bridges formed by H80/E132 and H95/E141 promote the rapid release of bound pheromone at an acidic pH; however, deprotonation of the H80 and H95 imidazole side-chains is expected to abolish the two salt bridges and contribute to pheromone binding (Xu et al., 2010). In the present study, many hydrophilic residues, including His75, were predicted to be located in the binding cavity as putative binding sites of AlinOBP13. Deprotonation of these hydrophilic residues, such as His75, may reasonably explain the considerably higher binding of AlinOBP13 to terpenoids at alkaline pH. Meanwhile, structural studies on the interaction of B. mori BmorGOBP2 complexed with sex pheromones and their analogues showed no involvement of its C-terminal (Zhou et al., 2009). A recent study in H. armigera OBP has also revealed better binding at acidic pH (Li et al., 2013). Considering the sequence and functional diversity of insect OBPs among different orders,

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Figure 10. Dose-dependent electroantennogram (EAG) responses of both female and male adults of Adelphocoris lineolatus to some terpenoids studied in the binding assays. Each sample was tested three times against at least six insects. EAG amplitudes (mean ± SE) are control-adjusted and presented as relative responses to the standard 10−1 3-hexanone. Each compound had different concentrations, including 10−4, 10−3, 10−2 and 10−1 (v/v), respectively. The standard error is represented by the error bar. Asterisks indicate significant differences between female and male (Student’s t-test): *P < 0.05, **P < 0.01 and ***P < 0.001.

© 2014 The Royal Entomological Society, 23, 417–434

Immunolocalization and binding of AlinOBP13 different mechanisms of ligand binding and release seem reasonable. So far, the actual pH in the sensillum lymph of Hemipteran species and whether or not the high binding affinities of AlinOBP13 to its ligands at alkaline pH has significant physiological relevance in A. lineolatus remain to be determined in further studies.

Experimental procedures Insect rearing and collection Adelphocoris lineolatus adults were collected from alfalfa fields at the Langfang Experimental Station of Chinese Academy of Agricultural Sciences, Hebei Province, China. The laboratory colony was established in accordance with the method described by Lu et al. (2008). The adults and newly emerged nymphs were reared on green beans and 10% honey in plastic containers (20 cm × 13 cm × 8 cm), which were maintained at 29 ± 1 °C, 60 ± 5% relative humidity, and 14h light:10 h dark cycle. Emerged adults (male: female = 1:1) and nymphs at different developmental stages were collected. The antennae of newly emerged female and male adults which had been starved or unstarved (acted as control) were collected after 4, 8 and 12 h of starving. All specimens were collected in triplicate, immediately frozen in liquid nitrogen, and either stored at −80 °C or used directly.

RNA extraction and cDNA synthesis Total RNA was isolated using the SV Total RNA Isolation System (Promega, Madison, WI, USA) following the manufacturer’s instructions. RNA quality was checked with a spectrophotometer (NanoDropTM1000, Thermo Fisher Scientific, Waltham, MA, USA) and 1.0% agarose gel electrophoresis. First-strand cDNA was synthesized using the SuperScriptTM III Reverse Transcriptase system (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions and immediately used for PCR amplification or stored at −20 °C until further use.

Quantitative revserse-transcriptase-PCR The expression of AlinOBP13 during the different developmental stages and the effect of starvation on AlinOBP13 expression were analysed through qRT-PCR on a 7500 Fast Detection System (Applied Biosystems, Carlsbad, CA, USA) using Taqman primers and probes designed using PRIMER EXPRESS 3.0 [Applied Biosystems (Table S1)]. The β-actin gene (GenBank No.GQ477013) of A. lineolatus was used as the internal control to normalize target gene expression and correct for sample-tosample variation. Each amplification reaction was performed in a 25 μl reaction volume containing 12.5 μl of remix ExTaq (TaKaRa, Kyoto, Japan), 1 μl of each primer (10 mM), 0.5 μl probe (10 mM), 0.5 μl Rox Reference Dye II, 1 μl sample cDNA and 8.5 μl sterilized H2O. The PCR reactions were run at 95 °C for 2 min, followed by 40 cycles of 95 °C for 20 s and 60 °C for 34 s. To check for authenticity, the qRT-PCR reaction of each sample was performed in three technical replicates and two biological replicates. The transcript levels of AlinOBP13 within the different developmental stages and at starvation intervals were analysed using the comparative 2−ΔΔCt method (Livak & Schmittgen, 2001). © 2014 The Royal Entomological Society, 23, 417–434

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Heterologous expression and purification of recombinant protein Gene-specific primers with restriction enzyme sites Nco I in the sense primer and Xho I in the antisense primer (Table S1) were used to clone cDNAs encoding for mature AlinOBP13. The reaction conditions were 94 °C for 4 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min, and a final 10 min elongation step at 72 °C. The correct product confirmed by sequencing was sub-cloned into the bacterial expression vector pET30a (+) (Novagen, Madison, WI, USA) previously digested with the same restriction enzymes. Plasmid contained valid insert was transformed into Escherichia coli BL21 (DE3)competent cells. A verified single colony was grown overnight in 5 ml of Luria-Bertani broth with 100 mg/ml kanamycin. The culture was diluted to 1:100 with fresh medium, and the growth was continued at 37 °C for 2 h to 3 h until an optical density (OD)600 value of 0.6 was reached. The production of the recombinant protein was induced with 1 mM isopropyl β-D-1thiogalactopyranoside at 37 °C for 6 h. The bacterial cells were harvested by centrifugation at 7000 × g for 20 min, resuspended in lysis buffer (80 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 4% glycerol, pH 7.2, and 0.5 mM phenylmethanesulfonyl fluoride), and then sonicated in ice (10 s, 5 passes). After centrifugation at 16 000 g for 20 min, the recombinant AlinOBP13, which was found in inclusion bodies, was solubilized and refolded using a previously described method (Prestwich, 1993). Subsequently, the refolded protein was collected and purified by two rounds of Ni ion affinity chromatography (GE Healthcare, Little Chalfont, UK), and the His-tag was removed with recombinant enterokinase (Novagen). Highly purified proteins were desalted by extensive dialysis. The size and purity of the recombinant AlinOBP13 were verified by 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).

AlinOBP13 antiserum preparation Polyclonal antiserum against recombinant AlinOBP13 was obtained by injecting robust adult rabbits subcutaneously and intramuscularly with highly purified recombinant AlinOBP13. The recombinant protein was emulsified with an equal volume of Freund’s complete adjuvant (Sigma, St. Louis, MO, USA) for the first injection (500 μg of recombinant protein) and incomplete adjuvant for further three additional injections (300 μg each time). The interval between each injection was 2 weeks. Blood was collected 7 days after the last injection and centrifuged at 6000 g for 20 min. The serum was finally purified using MAb Trap kit (GE Healthcare) following the manufacturer’s instructions. The rabbits were kept in large cages at room temperature. All operations were performed according to ethical guidelines to minimize pain and discomfort to the animals.

Western blot verification Crude male and female antennal extracts and purified recombinant AlinOBP13 were separated on 15% SDS-PAGE and subsequently transferred to a polyvinylidene fluoride membrane (Millipore, Carrigtwohill, Ireland). The membrane was blocked with 5% dry skimmed milk (BD Biosciences, San Jose, CA, USA) in phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBST) for 2 h at room temperature. After washing thrice with

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PBST (10 min each time), the blocked membrane was incubated with the purified rabbit anti-AlinOBP13 antiserum at 1:2500 dilution for 1 h. After washing three times with PBST again, the membrane was incubated with anti-rabbit IgG horseradish peroxidase conjugate and horseradish peroxidase–streptavidin complex (Promega) at 1:10 000 dilution for 1 h. After repeated washing, the membrane was incubated with Enhanced Chemiluminescence Western blot kit (CoWinbiotech, Beijing, China), and the bands were exposed to X-OMATBT films (Kodak, New York, NY, USA).

Transmission electron microscopy The antennae of newly emerged male and female adults were cut and fixed with a mixture of paraformaldehyde (4%), glutaraldehyde (2%), and 5% sucrose in 0.1 M PBS (pH 7.4) for 24 h at room temperature. Before post-fixing with 2% OsO4 in 0.1 M PBS (pH 7.4), the samples were rinsed five times with 0.1 M PBS (pH 7.4) for 30 min each, followed by dehydration in an ethanol series. The fixed antennae were then embedded in Epon 812 using propylene oxide. Ultrathin sections were cut with a glass knife on a LKB V Ultramicrotome (LKB Company, Bromma, Sweden) and then mounted on Formvar-coated grids. The sections were then observed using HITACHI H-7500 TEM (Hitachi Ltd, Tokyo, Japan).

Immunocytochemical localization Intact antennae detached from male and female adults were chemically fixed in a mixture of paraformaldehyde (4 %) and glutaraldehyde (2 %) in 0.1 M PBS (pH 7.4) at room temperature for 24 h, dehydrated in an ethanol series, and then embedded in LRWhite resin (Taab, Aldermaston, UK) for polymerization at 60 °C. Ultrathin sections (60 nm to 80 nm) were cut with a glass knife on a RMC MT-XL or with a diamond knife on a Reichert Ultracut ultramicrotome (Reichert Co., Vienna, Austria). For immunocytochemistry, the grids were subsequently floated, each time for 5 min, on 25 μl droplets of PBS (containing 50 mM glycine) and PBGT (PBS containing 0.2% gelatine, 1% bovine serum albumin, and 0.02% Tween-20). The grids were then incubated with primary antiserum diluted at 1:500 to 1:3000 with PBGT at 4 °C overnight. After washing six times with PBGT, secondary antibody (anti-rabbit IgG) coupled with 10 nm colloidal gold granules (Sigma) and diluted at 1:20 with PBGT was incubated with the sections at room temperature for 90 min. Optional silver intensification was used to enlarge the gold granules to approximately 40 nm, after which the sections were stained with 2% uranyl acetate to increase the contrast and observed in HITACHI H-7500 TEM (Hitachi Ltd). The serum supernatant from an uninjected healthy rabbit at the same dilution rate was used as the primary antiserum (negative control), and labelling experiments were conducted on three male and female adult antennae.

Fluorescence competitive binding assays Fluorescence binding assays were performed on an F-380 fluorescence spectrophotometer (Tianjin Gangdong Sci & Tech. Development. Co., Ltd, Tianjin, China) in a 1 cm light path quartz cuvette with 1 mM 1-NPN dissolved in methanol (Sigma) as a fluorescent probe. The slit width for both excitation and emission

was 10 nm. The excitation wavelength was 337 nm, and the emission spectrum was recorded between 390 and 460 nm. To measure the dissociation constant of recombinant AlinOBP13 to 1-NPN, a 2 μM solution of the protein in 50 mM Tris-HCl (pH 7.4) was titrated with aliquots of 1 mM 1-NPN dissolved in methanol to final concentrations ranging from 1 μM to16 μM. Forty chemical compounds were selected based on previous studies. The compounds were selected based on their presence either in host plants of A. lineolatus or in its sibling bugs (Aldrich, 1988; Loughrin et al., 1994; Pare & Tumlinson, 1997; Blackmer et al., 2004; Röse & Tumlinson, 2004; Table 1) to investigate the binding specificity of the recombinant AlinOBP13. Each chemical was dissolved in methanol at the final concentration of 1 mM. The affinity of each chemical was measured by competitive binding assays mixing AlinOBP13 and 1-NPN (both at 2 μM), followed by adding each ligand at the concentration range of 2–50 μM. Data on ligand binding were obtained from three independent experiments. The fluorescence intensities at the maximum fluorescence emission between 390 and 460 nm were plotted against free ligand concentration to determine binding constants. Bound chemical was evaluated based on fluorescence intensity with an assumption that the protein was 100% active with a stoichiometry of 1:1 (protein: ligand) saturation. The binding curves were linearized using a Scatchard plot, and the dissociation constants of competitors were calculated from the corresponding IC50 (ligand concentration displacing 50% of the fluorescence intensity of the AlinOBP13/1-NPN complex) values using the 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 1-NPN. The blue shift of 1-NPN was measured on the same equipment and parameters as the fluorescence competitive binding assay, except for a variation in the emission spectrum, which was between 390 nm and 560 nm. The fluorescence intensity of 5 μM 1-NPN in Tris-HCl buffer (pH 7.4) was measured with and without the recombinant AlinOBP13 at the final concentration of 5 μM. The effect of pH on the binding affinities was evaluated under three pH conditions using sodium acetate (pH 5.0) or Tris-HCl buffer (pH 7.4 and pH 10.0). Significant differences in binding affinities under the different pH conditions were analysed with ANOVA at a significance level of α = 0.05 using STATA 9.0 software (Stata Corp., College Station, TX, USA).

Electroantennogram recordings Newly emerged female and male adults were anaesthetized by chilling. The antenna was amputated at the base from the head. The tips of the antenna were removed to ensure good contact and then immediately attached to two electrode holders with nondrying clay (Spectra 360 Electrode Gel). The base of the antenna was connected to the reference electrode, and the tip of the antenna was connected to the recording electrode. An air stimulus controller CS-55 (Syntech, Kirchzarten, Germany) was used for air. Chemical stimulant delivery was performed at a constant flow of 10 ml/s. Signals were recorded for 5 s, beginning at 1 s before the onset of the stimulus pulse and passed through a high-impedance amplifier (CS-05 model; Syntech,). The EAG responses were initially measured in millivolts (peak height of depolarization) and then converted to normalized responses by the Syntech EAG 2000 program (Syntech). Eight terpenoids were tested in the dose-response EAG experiment with serial dilutions of each compound using paraffin oil (10−4, 10−3, 10−2, and 10−1 v/v; © 2014 The Royal Entomological Society, 23, 417–434

Immunolocalization and binding of AlinOBP13 Fluka, Buchs, Switzerland). 3-hexanone (10 μL at 10−1) was used as the reference compound to normalize all responses because it could elicit a stable EAG signal and no significant difference was observed between males and females. Subsequently, responses within an individual and among individuals were compared. The EAG responses were calculated by subtracting the mean response of control solvent and then converted to a percentage value of the mV response to the accompanying standard (10−1 3-hexanone) (Kendra et al., 2005). Each chemical (10 μL) was tested against six individual male and female antennae, with each antenna tested three times. All results are presented as normalized mean (± SE) EAG responses. EAG responses between male and female individuals were compared with the Student t-test using STATA 9.0 software.

Molecular modelling The amino acid sequence of AlinOBP13 was submitted to the platform of I-TASSER server for automated protein structure and function prediction (Roy et al., 2010). The fitness and validity between the sequence and the established sequence were evaluated using the 3D profile method (Lüthy et al., 1992) and the Ramachandran profile (Ramachandran et al., 1963). In addition, the function region and the binding site of the established 3D model were predicted based on the CASTp server (http:// sts.bioengr.uic.edu/castp/index.php).

Acknowledgements This work was supported by the China National ‘973’ Basic Research Program (2012CB114104) and the National Natural Science Foundation of China (31071694 and 31272048). Yong-Jun Zhang and Jing-Jiang Zhou acknowledge the financial support from the Royal Society, United Kingdom, for the international joint project between China and the United Kingdom (31111130203). References Aldrich, J.R. (1988) Chemical ecology of the Heteroptera. Annu Rev Entom 33: 211–238. Arimura, G.I., Matsui, K. and Takabayashi, J. (2009) Chemical and molecular mcology of herbivore-induced plant volatiles: proximate factors and their ultimate functions. Plant Cell Physiol 50: 911–923. Badonnel, K., Denis, J., Caillol, M., Monnerie, R., Piumi, F., Potier, M. et al. (2007) Transcription profile analysis reveals that OBP-1F mRNA is downregulated in the olfactory mucosa following food deprivation. Chem Senses 32: 697–710. Ban, L., Zhang, L., Yan, Y. and Pelosi, P. (2002) Binding properties of a locust’s chemosensory protein. Biochem Biophys Res Commun 293: 50–54. Bengtsson, M., Bäckman, A.C., Liblikas, I., Ramirez, M.I., Borg-Karlson, A.K., Ansebo, L. et al. (2001) Plant odor analysis of apple: antennal response of codling moth females to apple volatiles during phenological development. J Agr Food Chem 49: 3736–3741. Biessmann, H., Nguyen, Q., Le, D. and Walter, M. (2005) Microarray-based survey of a subset of putative olfactory

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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. Blue shift and increase in the intensity of fluorescence were observed when N-phenyl-1-naphthylamine (1-NPN) was bound to AlinOBP13. 1-NPN was dissolved in methanol and added to 50 mM Tris buffer (pH 7.4) at 5 μM, and the protein AlinOBP13 was also at 5 μM. When excited at 337 nm, 1-NPN in the Tris buffer produces a fluorescence peak with its maximum 450 nm, but in the presence of AlinOBP13, the emission wavelength is shifted to 405 nm accompanied by a sixfold increase in the intensity. Figure S2. Binding curves and Scatchard plots (insert) of the fluorescence probe N-phenyl-1-naphthylamine (1-NPN) to AlinOBP13. The dissociation constant of the AlinOBP13/1-NPN complex was 8.99 ± 0.90 μM. Figure S3. Binding curves and Scatchard plots for N-phenyl-1naphthylamine (1-NPN) to the recombinant AlinOBP13 at three pH values (5.0, 7.4 and 10.0).

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Figure S4. Effect of sodium ion concentrations (0, 30, and 60 mM) on the binding affinities of AlinOBP13 to β-caryophyllene and trans-β-farnesene at pH 7.4. Figure S5. Comparison of binding abilities (1/Ki) of two other insect OBPs with their ligands at pH 7.4 and 10.0. Binding activity of AlinOBP11 to nerolidol and binding activity of HarmPBP1 to Z11–16:ALD.

Figure S6. Predicted three-dimensional mode of AlinOBP13. Helices, N-terminal (N), and C-terminal (C) were labelled, and disulphide bridges were labelled yellow. Some hydrophobic and hydrophilic residues predicted as putative binding sites were displayed with green sticks. Table S1. Primers used in quantitative reverse transcriptase PCR and recombinant AlinOBP13 prokaryotic expression.

© 2014 The Royal Entomological Society, 23, 417–434