Insect Molecular Biology

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Insect Molecular Biology (2014) 23(6), 733–742

doi: 10.1111/imb.12119

The sensory neurone membrane protein SNMP1 contributes to the sensitivity of a pheromone detection system

Institute of Physiology, University of Hohenheim, Stuttgart, Germany

Keywords: olfaction, moth, pheromone detection, sensilla trichodea, sensory neurone membrane protein.

Abstract

Introduction

Male moths detect female-released sex pheromones with extraordinary sensitivity. The remarkable sensory ability is based on a cooperative interplay of pheromone binding proteins in the lymph of hair-like sensilla trichodea and pheromone receptors in the dendrites of sensory neurones. Here we examined whether in Heliothis virescens the so-called ‘sensory neurone membrane protein 1’ (SNMP1) may contribute to responsiveness to the pheromone component, (Z)-11-hexadecenal (Z11-16:Ald). By means of immunohistochemistry and in situ hybridization we demonstrated that SNMP1 is in fact present in cells expressing the Z11-16:Ald receptor HR13 and the dendrites of sensory neurones. To assess a possible function of SNMP1 we monitored the responsiveness of cell lines that expressed HR13 alone or the combination SNMP1/HR13 to stimulation with Z11-16:Ald by calcium imaging. It was found that SNMP1/HR13 cells were 1000-fold more sensitive to pheromone stimulation compared with HR13 cells. In contrast, cells that expressed HR13 and the non-neuronal SNMP2-type showed no change in pheromone sensitivity. Overall, our reconstitution experiments demonstrate that the presence of SNMP1 significantly increases the HR13based responsiveness of cells to Z11-16:Ald, suggesting that SNMP1 also contributes to the response of the antennal neurones and thus to the remarkable sensitivity of the pheromone detection system.

The highly sensitive detection of female-released pheromones by male moths has fascinated scientists for more than a century (Fabre, 1879; Schneider, 1992); however, the molecular basis for this extraordinary sensory capability is only partly understood (Stengl, 2010; Zhou, 2010; Leal, 2013; Sakurai et al., 2014). On the male antenna, the detection of pheromones involves pheromone-binding proteins (PBPs) in the aqueous lymph of hair-like structures, named sensilla trichodea, and pheromone receptors (PRs) residing in the membrane of sensory neurones, which extend their dendrites into the sensillum lymph (Blomquist & Vogt, 2003). The so-called ‘sensory neurone membrane proteins’ (SNMPs) have been identified as further characteristic proteins in pheromone-responsive sensilla of moths and other insects (Nichols & Vogt, 2008; Vogt et al., 2009). They are members of the CD36 (cluster of differentiation 36) superfamily, which comprises proteins involved in binding and translocation of lipophilic ligands or lipid−protein complexes (Martin et al., 2011; Valacchi et al., 2011). However, because of its localization in the dendritic membrane of pheromone-responsive neurones (Rogers et al., 2001b) and its co-expression with pheromone receptors (Benton et al., 2007; Forstner et al., 2008), it has been proposed that SNMP1 might function as a co-receptor or serve as a ‘docking-site’ for pheromone-loaded PBP, thereby helping to unload the pheromone from the PBP or to pass the signal molecules to the pheromone receptor (Rogers et al., 1997; Vogt, 2003). In several moth species a second SNMP-type (SNMP2) has been identified, which shares about 30% sequence identity with SNMP1 (Rogers et al., 2001a; Forstner et al., 2008; Gu et al., 2013; Liu et al., 2013; Zhang et al., 2014). Interestingly, in situ hybridization studies have shown that SNMP2 is expressed in supporting cells associated with the sensory

P. Pregitzer, M. Greschista, H. Breer and J. Krieger

First published online 21 July 2014. Correspondence: Dr Jürgen Krieger, Institute of Physiology (230), University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart, Germany. Tel.: +49 711 459 22265; e-mail: [email protected]

© 2014 The Royal Entomological Society

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neurones in pheromone-responsive sensilla (Forstner et al., 2008; Zhang et al., 2014). Based on the expression in supporting cells and the functional implication of CD36 proteins in translocation of lipophilic compounds (Martin et al., 2011; Valacchi et al., 2011), it has been proposed that SNMP2 may play a role in clearance processes within pheromone-responsive sensilla. In Drosophila melanogaster mutant flies a functional role of SNMP1 in the pheromone-responsive cells has been substantiated by electrophysiological analysis. It was found that in the absence of SNMP1, the Or67d (Odorant receptor 67d) neurones lack their sensitivity to the pheromone (Z)-11-vaccenyl acetate (Benton et al., 2007; Jin et al., 2008). Subsequently, protein complementation assays (Benton et al., 2007) and fluorescence resonance energy transfer studies (German et al., 2013) have indicated that SNMP1 is closely apposed to, although not necessarily directly interacting with, receptor proteins in the membrane. For the noctuid moth Heliothis virescens, there is compelling evidence that the detection of the major sex pheromone component (Z)-11-hexadecenal (Z11-16:Ald) by sensory neurones in long sensilla trichodea on the antenna (Almaas & Mustaparta, 1990; Berg et al., 1995; Baker et al., 2004) is based on the Heliothis virescens receptor 13 (HR13) (Krieger et al., 2004; Kurtovic et al., 2007; Wang et al., 2011; Vasquez et al., 2013) and involves the pheromone binding protein PBP2 (Krieger et al., 1993; Grosse-Wilde et al., 2007). Furthermore, reconstitution of the antennal PBP2/HR13 system in cell culture has provided evidence that both proteins contribute to the overall sensitivity of the response to pheromones (Grosse-Wilde et al., 2007). Ectopic expression of the H. virescens receptor HR13 in D. melanogaster Or67d neurones revealed that neuronal responses elicited by activation of HR13 with its ligand Z11-16:Ald are almost completely abolished in the absence of SNMP1 (Benton et al., 2007). This observation supports the notion that in male H. virescens antenna, the SNMP1 protein may contribute to the sensitivity of the HR13 neurones. In order to assess this possibility, in the present study we revisited the co-expression of SNMP1 and HR13 on the antenna of H. virescens. Furthermore, we generated cell lines that express HR13 alone or in combination with either HvirSNMP1 or HvirSNMP2 and monitored the responsiveness of the cells by calcium imaging experiments. Results To evaluate the role of SNMP1 in the HR13-mediated response to the major sex pheromone component, we reanalysed the expression of SNMP1 and HR13 in male antenna. By means of a specific antiserum and immuno-

histochemical approaches the SNMP1 protein was visualized in the somata and dendrites of individual cells located in long sensilla trichodea (Fig. 1A, B). This topographic distribution pattern resembles that of the HR13 receptor protein as shown in previous studies (Gohl & Krieger, 2006). As the antibodies for SNMP1 and HR13 were both generated in the same species, fluorescence in situ hybridization (FISH) in combination with fluorescence immunohistochemistry (FIHC) were used to visualize cells with SNMP1 and HR13. Using a HR13-specific antisense RNA probe and the SNMP1 antibody, we found cells that were labelled by both probes (Fig. 1C–E). In addition, cells that were positive only for SNMP1 were visible, suggesting co-expression of SNMP1 with other pheromone receptors. Together, the new and previous data clearly indicate that SNMP1 and HR13 are localized in the somata and dendrites of the same cells. To assess a possible role of SNMP1 in pheromone detection, we generated stable cell lines carrying the coding sequence for HR13 or both SNMP1 and HR13 (Fig. 2A, B, left). For co-expression of HR13 and SNMPs in the same cell we integrated a bicystronic expression cassette into the genome of T-Rex293/Gα15 cells. The expression of the integrated genes was verified using reverse transcription PCR (RT-PCR) and primer pairs matching the coding sequences of SNMP1 or HR13 (Fig. 2A, B, right). From cDNA of the HR13-expressing cells a PCR product of the correct size was amplified with HR13 primers, whereas SNMP1- and SNMP2specific primers (controls) gave no amplification products. From cDNA of SNMP1/HR13-expressing cells bands of the predicted size were amplified with SNMP1and HR13-specific primers, but not with primers for SNMP2. Approaches without reverse transcriptase underscored the specificity of the PCR results and ruled out amplification from genomic DNA. Together, the results of the RT-PCR experiments confirmed the correct genome insertion of the HR13 and SNMP1/HR13 expression cassettes and transcription of the pheromone receptor and SNMP1 coding regions in the corresponding cells. To monitor the response of HR13-expressing cells to the major sex pheromone, we performed Fura-2-based calcium imaging experiments. It was found that stimulation of HR13 cells with 10 nM Z11-16:Ald solubilized by means of dimethylsulphoxide (DMSO) elicited a significant transient shaped calcium response of the cells (Fig. 3, top). Dose response experiments indicated a concentration-dependent responsiveness of the cells (Fig. 3, bottom). These results demonstrate that expression of the HR13 receptor protein renders the T-Rex293/ Gα15 cells responsive to Z11-16:Ald, thus confirming and extending previous observations (Grosse-Wilde et al., 2007; Pregitzer et al., 2012). © 2014 The Royal Entomological Society, 23, 733–742

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Figure 1. Co-expression of sensory neurone membrane protein 1 (SNMP1) and Heliothis virescens receptor 13 (HR13) in the male antenna. (A, B) Cryosection of male Heliothis virescens antenna probed with an antibody specific for SNMP1. Immunoreactivity was visualized by an Alexa488 secondary antibody. Two SNMP1-expressing cells are shown and can be assigned to long sensilla trichodea. Immunoreactivity is visible in the somata and the dendrites of SNMP1-positive cells. The green fluorescence channel (B) was overlaid with the transmitted light channel in (A). (C−E) Combined fluorescence in situ hybridization and immunohistochemistry using an SNMP1 antibody (green) and a digoxigenin-labelled antisense RNA probe for HR13 (red). The red and green fluorescence channels are shown separately in (D) and (E). All HR13-positive cells also expressed SNMP1. Scale bars = 20 μM.

To investigate whether co-expression of SNMP1 may affect the responsiveness of HR13-expressing cells we next performed calcium imaging experiments with T-Rex293/Gα15 cells expressing SNMP1 and HR13. Application of a strong pheromone stimulus (10 nM) to SNMP1/HR13 cells (Fig. 4, top) resulted in a calcium response quite similar to HR13-expressing cells. However, when the threshold pheromone dose for HR13 cells (1 pM) was applied to SNMP1/HR13-expressing cells, they still gave a maximal response (Fig. 4, middle). Extending the dose response experiments to even lower Z11-16:Ald concentrations resulted in significant responses of SNMP1/HR13 cells down to 1 fM pheromone (Fig. 4, bottom). Comparing the calcium responses of cells expressing SNMP1 and HR13 with cells having only HR13 (Fig. 4, bottom) revealed that the SNMP1/ HR13-expressing cells were apparently ∼1000-fold more sensitive to pheromone stimulation. To further support the notion that the increased sensitivity of SNMP1/HR13-expressing cells was due to the © 2014 The Royal Entomological Society, 23, 733–742

presence of the olfactory sensory neuron (OSN)-specific SNMP1 type (Forstner et al., 2008), we performed similar experiments with HR13 cells co-expressing the related subtype SNMP2. SNMP2 shares sequence similarity and some structural features with SNMP1 but is expressed in supporting cells surrounding the HR13expressing OSNs in pheromone-responsive sensilla (Forstner et al., 2008). For co-expression of SNMP2 and HR13 in T-Rex293/Gα15 cells we generated a cell line carrying a bicystronic expression vector construct (Fig. 2C, left). From the cDNA of SNMP2/HR13expressing cells, bands of the correct size were amplified by RT-PCR with primers specific to SNMP2 or HR13 (Fig. 2C, right), confirming correct gene integration and transcription. Stimulation of SNMP2/HR13-expressing cells with 10 nM Z11-16:Ald pheromone (Fig. 5, top) elicited a significant calcium response, indicating that the cells express a functional pheromone receptor. However, comparing the response of SNMP2/HR13 cells with the corresponding responses of HR13 cells and SNMP1/

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Figure 2. Expression of HR13, sensory neurone membrane protein 1 (SNMP1) and SNMP2 in T-Rex293/Gα15 cells. (A–C) Left: constructs for generating stable cell lines, encoding either for HR13 (purple), SNMP1 (red) and HR13 or SNMP2 (green) and HR13. (A−C) Right: reverse transcription PCR (RT-PCR) with cDNA prepared from the resulting cell lines and specific primers for SNMP1 (S1), SNMP2 (S2), HR13 (R13). (A) A band of the expected size indicates expression of HR13, but not for SNMP1 and SNMP2 in the cell line transfected with the HR13-expressing construct. (B) In the cell line transfected with the SNMP1/HR13 construct a band could only be amplified with SNMP1 and HR13 primers. (C) In the cell line transfected with the SNMP2/HR13 construct, bands of the right size were obtained with primer pairs for SNMP2 and HR13 but not for SNMP1. No amplification products were obtained in RT-PCR approaches without reverse transcriptase. IRES, internal ribosome entry site.

HR13 cells indicated that the response was generally lower in SNMP2/HR13 cells (Fig. 5, bottom). A significant calcium response was only obtained at a concentration of 100 pM, compared with 1 pM and 1 fM of HR13 and SNMP1/HR13, respectively. These results show that SNMP2/HR13-expressing cells are less sensitive to

Z11-16:Ald than HR13-expressing cells and much less than SNMP1/HR13-expressing cells. Moreover, the results indicate that SNMP2 cannot functionally replace SNMP1 and suggest that SNMP1 may play a specific role in the highly sensitive response to hydrophobic pheromone ligands.

Figure 3. Response of HR13-expressing cells to (Z)-11-hexadecenal (Z11-16:Ald). Top: pseudocolour images indicate calcium levels in HR13-expressing cells after stimulation with Ringer solution containing 0.1% dimethylsulphoxide, 0.1% n-hexane (control) or the same solution containing 10 nM Z11-16:Ald. The colour bar indicates low (L) to high (H) calcium concentration as blue to red changes, respectively. The changes in intracellular calcium in a representative cell from the experiment are shown to the right as changes of Fura-2 fluorescence intensity ratios (340/380 nm) over time. C, control; S, Z11-16:Ald stimulus. Bottom: responses of HR13-expressing cells to stimulation with different concentrations of Z11-16:Ald. Bars represent the mean responses of cells reported as F/F0 ± SE ratios determined from three to seven independent replicates with at least 30 cells in each experiment. Responses that differed significantly from those of the control are indicated by asterisks: * P < 0.05, *** P < 0.001 (one-way ANOVA followed by Dunnett’s post test).

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Figure 4. Response of SNMP1/HR13-expressing cells to (Z)-11-hexadecenal (Z11-16:Ald). Top, left and middle: pseudocolour images indicating calcium levels in SNMP1/HR13-expressing cells after application of a control stimulus (Ringer with 0.1% dimethylsulphoxide, 0.1% n-hexane) or stimulation with solutions containing 10 nM and 1 pM Z11-16:Ald, respectively. The colour bar indicates low (L) to high (H) calcium concentration as blue to red changes, respectively. Top, right: calcium responses of representative cells from the experiment monitored as changes of Fura-2 fluorescence intensity ratios (340/ 380 nm) over time. C, control; S, Z11-16:Ald stimulus. Bottom: responses of SNMP1/HR13-expressing cells to stimulation with different concentrations of Z11-16:Ald (striped bars). For comparison, the responses of HR13-expressing cells from Fig. 3 are shown as grey bars. Bars represent the mean responses of cells reported as F/F0 ± SE ratios determined from three to seven independent replicates with at least 30 cells in each experiment. Responses that differed significantly from those of the control (one-way ANOVA followed by Dunnett’s post test) are indicated by asterisks: * P < 0.05, *** P < 0.001.

Discussion The results of the present study support the concept that SNMP1 may contribute to the sensitive responsiveness of male H. virescens to the female-released sex-pheromone component, Z11-16:Ald. The visualization of the SNMP1 protein in cell bodies and dendrites of sensory neurones within the long trichoid sensilla of H. virescens, which are responsive to pheromone components (Almaas & Mustaparta, 1991; Baker et al., 2004), confirms and © 2014 The Royal Entomological Society, 23, 733–742

extends the original findings on SNMP1 localization in male antennae of the moth Antheraea polyphemus (Rogers et al., 1997, 2001b). In addition, it has been shown that SNMP1 is also expressed in OSNs on the antennae of female moths (Rogers et al., 2001a, 2001b; Gu et al., 2013; Zhang et al., 2014) but the identity of these OSNs is unknown. We determined the identity of some SNMP1-positive cells in H. virescens males by a combined immunohistochemical and in situ hybridization approach. The results

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Figure 5. Responses of SNMP2/HR13-expressing cells to (Z)-11-hexadecenal (Z11-16:Ald). Top, left and middle: calcium levels in SNMP2/HR13expressing cells after stimulation with Ringer solution containing 0.1% dimethylsulphoxide, 0.1% n-hexane (control) or the same solution containing 10 nM Z11-16:Ald presented as pseudocolour images. The colour bar indicates low (L) to high (H) calcium concentration as blue to red changes, respectively. Top, right: changes in intracellular calcium responses over time of a representative cell from the experiment shown as changes of Fura-2 fluorescence intensity ratios (340/380 nm). C, control; S, Z11-16:Ald stimulus. Bottom: responses of SNMP2/HR13-expressing cells to stimulation with different concentrations of Z11-16:Ald (striped bars). For comparison the responses of HR13-expressing cells are shown (grey bars). Bars represent the mean responses of cells reported as F/F0 ± SE ratios determined from three to four independent replicates with at least 30 cells in each experiment. One-way ANOVA followed by Dunnett’s post test was used to verify responses that differed significantly from those of the control. * P < 0.05, ** P < 0.01, *** P < 0.001.

indicate that cells that express the pheromone receptor HR13 also express SNMP1 (Fig. 1). Moreover, SNMP1 was located in the dendrites of neurones in long sensilla trichodea; interestingly, in a previous study we showed that HR13 receptor protein is also found in the dendrites (Gohl & Krieger, 2006). Thus, the receptor protein for the main pheromone component Z11-16:Ald and the SNMP1 protein are apparently co-localized in the chemosensory dendritic membrane of the neurones. Whether HvirSNMP1 and the receptor HR13 are structurally interacting or form heteromeric complexes in the membrane as suggested for the pheromone-responsive cells in D. melanogaster (Jin et al., 2008) remains elusive. The possible interaction of SNMP1 and/or the pheromone receptor protein with the olfactory co-receptor (Orco) as proposed for the fly system (Ha & Smith, 2009) also remains unclear in moths. Although Orco is expressed in antennal neurones of moths (Sakurai et al., 2004; Mitsuno et al., 2008), its contribution to the primary processes of pheromone reception is still under debate (Stengl, 2010; Stengl & Funk, 2013; Sakurai et al., 2014). Consistent with our previous results (Grosse-Wilde et al., 2007; Pregitzer et al., 2012), in imaging experiments it was possible to monitor the pheromone-induced responses of T-Rex293/Gα15 cells expressing the HR13 receptor in the absence of Orco. These observations indicate that in the heterologous expression system the activation of the HR13 receptor does not require Orco. The dose-dependent response experiments exploring the effect of SNMP1 on the Z11-

16:Ald responsiveness of HR13-expressing cells revealed that in the presence of SNMP1 the sensitivity was significantly increased. Whereas the threshold concentrations for HR13 cells was determined to be between 1 pM (this study) and 10 pM (Grosse-Wilde et al., 2007), the threshold concentration determined for SNMP1/HR13 cells was about 1 femtomolar. Thus, in the presence of SNMP1 the responsiveness of the cells was increased by about 1000-fold. The higher sensitivity to Z11-16:Ald was found in HR13/ SNMP1 co-expressing cells but not in HR13 cells co-expressing SNMP2. This result indicates that SNMP2 cannot functionally substitute SNMP1. This functional difference between SNMP1 and SNMP2 is in line with their different cellular expression; whereas SNMP1 is exclusively found in olfactory neurones, SNMP2 is found in supporting cells (Forstner et al., 2008; Zhang et al., 2014). Although it cannot be excluded that an increase in sensitivity of the HR13/SNMP1-expressing cells could be because of higher levels of HR13 receptor protein in the cells, the RT-PCR experiments indicate similar or if anything lower levels of mRNA for the HR13 receptor in SNMP1/HR13-cells compared with HR13-cells or HR13/ SNMP2-cells. How the SNMP1 protein may contribute to increased sensitivity of the HR13-expressing cells remains elusive. SNMP1 is apparently not required for the targeting of pheromone receptors to the membrane (Benton et al., 2007); however, it has been speculated that SNMP1 may contribute to stabilizing the receptor protein in a conformation that is more accessible for the pheromones. It has also © 2014 The Royal Entomological Society, 23, 733–742

Function of SNMP1 in pheromone detection been proposed that the extended extracellular loop of the SNMP1 protein may be involved in ‘capturing’ the pheromone molecules or in an efficient transfer of the pheromone to the binding site of the receptor. SNMP1 has been noticed to share several structural features with proteins of the CD36 family, including the membrane topology with a long extracellular domain (Rogers et al., 1997; Nichols & Vogt, 2008). As CD36 proteins interact with lipoproteins and contribute to the transfer of the bound lipophilic compounds, it has been speculated that SNMP1 may serve as a ‘docking site’ for pheromone-loaded PBP and as a supporting structure for channelling the pheromone to the receptor (Vogt 2003; Benton et al., 2007; Forstner et al., 2008). So far, there is no experimental support for this concept. Studies with cell lines expressing receptors have shown that the presence of a distinct PBP also contributes to an increased sensitivity of the detection system for a pheromone component (Grosse-Wilde et al., 2007; Forstner et al., 2009). Future reconstitution approaches employing PBP2, the binding protein for Z11-16:Ald (Grosse-Wilde et al., 2007), in experiments with SNMP1/HR13expressing cells, may clarify the specific contribution of each SNMP1 and PBP to the sensitivity of pheromone detection. Together, the results provide evidence that in H. virescens, not only the pheromone receptor and the binding protein, but also SNMP1 contributes to the extreme sensitivity of pheromone-responsive antennal neurones. Experimental procedures

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room temperature in a humid box. Finally, slides were washed three times for 5 min each with PBS and mounted in mowiol solution (13% mowiol 4–88, 33% glycerine, 130 mM Tris, pH 8.5). Combined in situ hybridization and immunohistochemistry Combined FISH and FIHC were performed on sections using protocols described earlier (Gohl & Krieger, 2006). Briefly, cryosections (prepared as described under Immunohistochemistry) were rinsed in PBS, fixed for 30 min at 4 °C using 4% paraformaldehyde in phosphate buffer with 1% Triton X-100 and washed at room temperature for 1 min each in PBS and PBS with 1% Triton X-100. In all subsequent incubation steps slides were placed in a humid box containing filter paper soaked with 50% formamide (for hybridization) or H2O (all other steps). Slides were incubated for 10 min in 50% formamide, 5 × SSC (sodium chloride/sodium citrate) (1 × SSC = 0.15 M NaCl, 0.015 M Na-citrate, pH 7.0) at 4 °C, followed by incubation with a digoxigenin-labelled HR13 antisense RNA probe (Krieger et al., 2004) diluted in hybridization buffer at 55 °C overnight. Posthybridization, sections were washed twice for 30 min in 0.1 × SSC at 60 °C, then treated for 30 min with blocking solution (PBS with 10% normal goat serum, 0.3% Triton X-100) and incubated overnight at 8 °C with the primary antibody (antiApolSNMP1 antiserum, 1:750 in blocking solution). After three washes for 5 min each in PBS, the primary antibody and the digoxigenin-labelled probe were detected by incubating the slides for 2 h at room temperature with an antirabbit Alexa 488 antibody (1:500; Molecular Probes Europe) and an antidigoxigenin alkaline phosphatase-conjugated antibody (1:1000; Roche, Mannheim, Germany), respectively, in blocking solution. After three 5 min washes with PBS, in situ hybridization signals were visualized using the HNPP (2-hydroxy-3-naphtoic acid-2′-phenylanilide phosphate)/Fast Red fluorescence detection set (Roche) as described earlier (Krieger et al., 2002, 2004). After three final washes for 5 min each with PBS, sections were embedded in mowiol solution.

Immunohistochemistry Immunohistochemistry was performed as described previously (Gohl & Krieger, 2006) with few modifications. Antennae of 1- to 2-day-old male H. virescens moths were dissected and fixed for 1 h in 4% paraformaldehyde in phosphate buffer (1.4 mM KH2PO4, 8 mM Na2HPO4, pH 7.4) with 1% Triton X-100. After fixation the tissue was briefly rinsed in phosphate-buffered saline (PBS; 0.85% NaCl, 1.4 mM KH2PO4, 8 mM Na2HPO4, pH 7.4) and incubated in 25% sucrose in PBS overnight at 8 °C. After rinsing in PBS antennae were embedded in O.C.T. (optimal cutting temperature compound) Tissue Tek freezing medium (Sakura Finetek Europe, Zoeterwoude, the Netherlands) and frozen at −22 °C. Cryosections (12 μm) of antennae were thawmounted on Superfrost slides (Menzel-Gläser, Braunschweig, Germany) and air-dried at room temperature for at least 30 min. After washing slides for 5 min in PBS, sections were covered with blocking solution (PBS with 10% normal goat serum, 0.3% Triton X-100) and incubated for 30 min at room temperature in a humid box. The blocking solution was replaced by solution containing the primary antibody [anti-Antheraea polyphemus SNMP1 (antiApolSNMP1) antiserum, 1:750 in blocking solution; Rogers et al., 1997] and sections were incubated overnight at 8 °C. After washing three times for 5 min in PBS, sections were treated with an antirabbit Alexa 488 antibody (1:500; Molecular Probes Europe, Leiden, the Netherlands; in blocking solution) for 2 h at © 2014 The Royal Entomological Society, 23, 733–742

Analysis of antennal sections by microscopy Sections from FIHC and FISH experiments were analysed on a LSM 510 meta laser scanning microscope (Zeiss, Oberkochen, Germany). Confocal image stacks of the fluorescence and transmitted-light channels were used to obtain projections of optical planes. In addition, pictures were generated in which the red and green fluorescence channels were overlaid with the transmitted light channel or shown separately. Generation of SNMP1/HR13- and SNMP2/HR13-expressing cell lines Cell lines with stable genome integration of the coding sequences for both SNMP1 and HR13, respectively SNMP2 and HR13, were generated using the components of the Flp-In-System (Invitrogen, Karlsruhe, Germany). We used Flp-In T-REx293/ Gα15 cells previously shown to be well suited for expression of functional moth pheromone receptors (including HR13) and to analyse receptor-mediated pheromone responses by Fura-2 based calcium-imaging (Grosse-Wilde et al., 2006, 2007; Forstner et al., 2009; Pregitzer et al., 2012). The generation of the construct for preparing HR13-expressing cells has been described earlier (Grosse-Wilde et al., 2007; Pregitzer et al., 2012). To generate Flp-In T-REx293/Gα15 cells,

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which co-express the pheromone receptor HR13 and SNMP1, a SNMP1/IRES (internal ribosome entry site)/HR13 expression cassette was generated in the pcDNA5-vector. First, the coding region of H. virescens SNMP1 was PCR-amplified from a plasmid carrying the SNMP1 cDNA sequence (Rogers et al., 2001a) using sense and antisense primers with restriction sites (underlined) added for NheI (5′-ATTTGCTAGCATGCAGCTGCCTAAGGA GCT-3′) and EcoRI (5′-CCGGAATTCCGATTATATGTTCACC TTAGC-3′), respectively. PCR conditions (if not otherwise specified) in this and all the following amplification steps were as described under ‘Reverse transcription PCR’ (see below). The PCR product was integrated into the corresponding restriction sites of the pIRES-vector (Clontech, Mountain View, CA, USA), followed by PCR amplification of the SNMP1/IRES DNA fragment using sequence-specific primers flanked with restriction sites for EcoRV (5′-AGTGATATCATGCAGCTGCCTAAGGAGCTA-3′) and NotI (5′-ATTTGCGGCCGCGTTGTGGCAAGCTTATCATCG-3′). In this case PCR conditions were: 4 min at 94 °C, then 30 cycles with 94 °C for 30 s, 65 °C for 40 s and 72 °C for 1 min 10 s, followed by incubation for 5 min at 72 °C. Using the EcoRV/NotI restriction sites the SNMP1/IRES DNA was cloned into the pcDNA5 plasmid (Invitrogen). Finally, the coding region of HR13 was PCR amplified from a plasmid (Krieger et al., 2005) using primers with added NotI (5′-ATTTGCGGCCGCGATGAAAATCCT ATCGGACGG-3′) and XhoI (5′-CCGCTCGAGATTTTATTCTTCT TCTGCAACTGT-3′) sites and cloned into the respective sites of pcDNA5/SNMP1/IRES, resulting in pcDNA5/SNMP1/IRES/ HR13. For the generation of pcDNA5/SNMP2/IRES/HR13 a different approach was used. First the IRES sequence was PCR amplified from the pIRES-vector and cloned into the pGEM-T vector (Promega, Mannheim, Germany) using primers that added the SacII/BamHI/EcoRV (5′-CCGCGGGGATCCGATATCAATTCCG CCCCTCTCCCCCCCC-3′) and SpeI/XhoI (5′-ACTAGTGAGCTC TTATCATCGTGTTTTTCAAAGGAAAACCACGTCCC-3′) restriction sites to IRES-pGEM-T. Next, the coding region of H. virescens SNMP2 was PCR amplified from a plasmid (Forstner et al., 2008) using a specific primer pair with BamHI (5′-ACTGAGGATCCATGTTGGGCAAACACTCGAAAATAT-3′) and EcoRV (5′-GGATTGATATCTCAATTTCCTTTATTAACCTG AGTA-3′) restriction sites. SNMP2 was cloned into IRES-pGEM-T using the BamHI and EcoRV sites. Subsequently, SNMP2/IRES was cut out with BamHI/NotI from SNMP2/IRES-pGEM-T and cloned into the respective sites of pcDNA5/HR13 (Grosse-Wilde et al., 2007), resulting in pcDNA5/SNMP2/IRES/HR13. The sequences of the expression cassettes in pcDNA5/SNMP1/IRES/ HR13 and pcDNA5/SNMP2/IRES/HR13 were checked for correct amplification and integration by sequencing on an ABI310 sequencing system using specific primers and a BIG dye cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). For generation of stable cell lines, the plasmids pcDNA5/ SNMP1/IRES/HR13, respectively pcDNA5/SNMP2/IRES/HR13, were transfected into T-Rex293/Gα15 cells using lipofectamine (Invitrogen) following the supplier’s protocol. 48 h after transfection, cells were selected for integration of expression constructs into the genome using media supplemented with 100 mg/L hygromycin. Genome integration of SNMP1, SNMP2 and HR13 was further verified by PCR using specific primers and genomic DNA, isolated from hygromycin-resistant SNMP1/HR13 or SNMP2/HR13 cells. Cell lines were cultured using DMEM (Dulbecco’s Modified Eagle’s Medium) media (Invitrogen) sup-

plemented with 10% foetal bovine serum (Invitrogen) and either 100 mg/L hygromycin, 10 mg/L blasticidin or 200 mg/L geneticin in regular alternation. RT-PCR Total RNA of cells was prepared from a T75 flask with 90% confluence using a NucleoSpin RNA Kit (Macherey-Nagel, Düren, Germany) following the recommendations of the supplier, but using a prolonged incubation time (1 h) with DNase. cDNA was transcribed from total RNA as previously described (Krieger et al., 2002) and used in RT-PCR experiments with the specific primer pairs SNMP1 (5′-GCTACGTCGCGAACATCGGAG-3′ and 5′-TA TGTTCACCTTAGCAGGCTC-3′), SNMP2 (5′-AGGACGCCTTC CTCAGGGTC-3′ and 5′-TTGGAGTCGCTTTACCCCAC-3′) and HR13 (5′-CGGTCTACTTACTCGGCTTGG-3′ and 5′-CTGTGCG ACTGTCTGAGCATC-3′). PCR conditions were 1 min 40 s at 94 °C, then 21 cycles at 94 °C for 30 s, 55 °C for 40 s and 72 °C for 1 min 30 s, with a decrease in the annealing temperature of 0.5 °C per cycle. Subsequently, 19 further cycles under the conditions of the last cycling step were performed, followed by incubation for 7 min at 72 °C. PCR products were analysed by agarose-gel electrophoresis. Calcium imaging of SNMP1/HR13-, SNMP2/HR13- and HR13-expressing cells Fura-2 was obtained from Invitrogen. Fura-2-based calcium imaging experiments were performed as described in detail previously (Grosse-Wilde et al., 2006, 2007; Forstner et al., 2009). (Z)-11-hexadecenal (Z11-16:Ald) was purchased from Fluka, München, Germany. Z11-16:Ald dilutions were made from stock solutions in n-hexane using Ringer solution (138 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.5 mM MgCl2, 10 mM Hepes, 10 mM Glucose, pH 7.3) with 0.1% DMSO. Pheromone dilutions were prepared freshly before the imaging experiments and were used within 2 h. Data analysis and acquisition was performed with the Metafluor imaging system and METAFLUOR 4 software (Visitron Systems, Puchheim, Germany). In order to monitor changes in [Ca2+]i concentration in single cells, the intensity of fluorescent light emission at 510 nm over time was measured, using excitation at 340 and 380 nm. The increase in the ratio of fluorescence emission at 340/380 nm excitation was used as the index for a rise in intracellular free calcium. To calculate the ratios the Fura-2 fluorescence intensity values of cells were taken before (F0) and after stimulation (F; peak of the response) with ligands. In a single experiment F/F0 values of at least 30 individual cells were determined and averaged.

Acknowledgements We are grateful to Prof. Richard Vogt (University of South Carolina) for kindly providing us with the anti-ApolSNMP1 antiserum. The authors would like to thank the insect rearing unit at Bayer CropScience AG Frankfurt for providing H. virescens pupae. Heidrun Froß is acknowledged for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, SPP1392 by grants to J. K. (KR1786/4-2). © 2014 The Royal Entomological Society, 23, 733–742

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© 2014 The Royal Entomological Society, 23, 733–742