Insect Science (2014) 00, 1–10, DOI 10.1111/1744-7917.12131
ORIGINAL ARTICLE
Identification and localization of two sensory neuron membrane proteins from Spodoptera litura (Lepidoptera: Noctuidae) Jin Zhang1,2 , Yang Liu1 , William B. Walker3 , Shuang-Lin Dong2 and Gui-Rong Wang1 1 State
Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural
Sciences, Beijing, 2 Education Ministry Key Laboratory of Integrated Management of Crop Disease and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing, China and 3 Swedish University of Agricultural Sciences, Department of Plant Protection Biology, Chemical Ecology Research Group, Alnarp, Sweden
Abstract Sensory neuron membrane proteins (SNMPs), which are located on the dendritic membrane of olfactory sensory neurons (OSNs), are proposed to be associated with odor reception in insects. Recent studies have demonstrated that SNMP1 is essential for electrophysiological responses of OSNs to the sex pheromone, cis-vaccenyl acetate (cVA) in Drosophila melanogaster. To investigate the function of Lepidoptera SNMPs, we cloned two SNMP genes, SlituSNMP1 and SltiuSNMP2, from Spodoptera litura (Lepidoptera: Noctuidae). Sequence alignment and phylogenetic analysis showed that both genes bear the general characteristics of SNMPs, including six conserved cysteine residues and two transmembrane domains. Further expression profile experiments showed that SlituSNMP1 is mainly expressed in the antenna, while SlituSNMP2 is broadly expressed in various tissues. By in situ hybridization experiments, it was found that SlituSNMP1 expressing cells are surrounded by the SlituSNMP2 expressing cells in the pheromone sensitive sensilla, suggesting different functions of the two SNMPs in insect olfaction. Key words In situ hybridization, Spodoptera litura, sensory neuron membrane proteins (SNMPs)
Introduction Sensory neuron membrane proteins (SNMPs), first identified in Lepidoptera insects, are transmembrane proteins localized in dendritic membrane of the pheromone-sensing olfactory receptor neurons (ORNs) and are proposed to play an important role in pheromone reception in insects (Rogers et al., 1997; Rogers et al., 2001b; Benton et al., 2007; Forstner et al., 2008; Jin et al., 2008). SNMPs belong to a gene family of the human fatty
Correspondence: Gui-Rong Wang, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China. Tel: +86 010 62816947, Fax: +86 010 62896114; Email: [email protected]
acid transporter (FAT), CD36, which is characterized by two transmembrane domains and is mainly involved in the recognition of fatty acids, cholesterol and proteinaceous compounds in cells (Rogers et al., 1997; Rasmussen et al., 1998; Levy et al., 2007; Nassir et al., 2007; Febbraio & Silverstein, 2007; Fukuwatari et al., 1997). In the last decade, two SNMP orthologues (SNMP1 and SNMP2) have been characterized in several insect orders, including Lepidoptera, Hymenoptera, Diptera, Coleoptera and so on (Nichols & Vogt, 2008; Rogers et al., 2001a; Vogt et al., 2009; Forstner et al., 2008). Studies on Drosophila melanogaster demonstrated that DmelSNMP1 is essential in detecting the fruit fly pheromone cis-vaccenyl acetate (cVA) (Benton et al., 2007; Jin et al., 2008). Additionally, DmelSNMP1 was also required for the activation of the ectopically expressed pheromone receptor HvirOR13 (Heliothis 1
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virescens) by (Z)-11-hexadecenal in OR67d-expressing neurons (Benton et al., 2007). Forstner et al. (2008) reported that HvirSNMP1 is coexpressed with HvirOR13 in the same ORNs in the antenna, but HvirSNMP2 is expressed in the supporting cells that surround the HvirOR13 neurons. A similar expression pattern was also found for ApolSNMP1 and ApolSNMP2 on the antennae of male Antheraea polyphemus (Forstner et al., 2008). Recently the SNMPs in Cnaphalocrocis medinalis, Agrotis ipsilon and S. exigua have been identified and discussed (Liu et al., 2013; Gu et al., 2013; Liu et al., 2014). The exact function of SNMPs in moths is still unknown, but the essential role in pheromone reception in D. melanogaster and the broad expression in pheromone sensing sensilla indicate they may be involved in pheromone detection in Lepidoptera. To elucidate whether the SNMP expression patterns are identical in Lepidoptera or distinct in some species, we identified two SNMPs (SlituSNMP1 and SlituSNMP2) from the common cutworm, Spodoptera litura, and investigated the expression patterns of the two genes regarding different tissues and genders, and finally, determined the topographic and cell-specific expression of the two SNMPs in male antennae.
open reading frame sequences, rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) was performed using SMARTerTM RACE cDNA amplification kit (Clontech, Mountain View, CA, USA) for 5’ and 3’ ends amplification. The full-length sequences were assembled based on reverse transcription PCR (RT-PCR) and RACE results and then confirmed by end-to-end PCR using specific primers designed at both ends. The primers are listed in Table S1. Bioinformatic analysis Homologous SNMP sequences were searched from GenBank. Transmembrane domains of SlituSNMPs were predicted with TMHMM (http://www. cbs.dtu.dk/services/TMHMM). The unrooted consensus neighbor-joining (NJ) tree was constructed using the NJ method and evaluated by the bootstrap procedure based on 1000 replicates with pairwise gap deletions in MEGA 5.0 software (The Biodesign Institute, Center for Evolutionary Functional Genomics, Tempe, AZ, USA). The theoretical isoelectric points (pI) and molecular weights of the deduced SlituSNMP1 and SlituSNMP2 were obtained by using ExPASy server software (http://www.expasy.org/tools/protparam.html).
Materials and methods Tissue expression pattern
Insects
To confirm the expression profiles of SlituSNMP1 and SlituSNMP2, antennae, proboscises, maxillary palps, legs and genitalia were isolated from male and female adults of S. litura. RNA extraction and cDNA synthesis were performed as described above. A “house-keeping” gene of S. litura encoding actin (DQ494753.1) was used to verify the integrity of the cDNA. The PCR programs were 95°C for 3 min, 35 cycles at 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s, with a final 10 min incubation at 72°C. PCR products were analyzed on 2.0% agarose gels. Quantitative RT-PCR (qRT-PCR) analysis was conducted on the ABI Prism 7500 Fast Detection System (Applied Biosystems, Carlsbad, CA, USA). Two reference genes GAPDH (GenBank No. HQ012003.2) and elongation factor 1-a (GenBank No. KC007373.1) were used to normalize the target gene expression and to correct sample-tosample variation. The qRT-PCR primers were designed using the Beacon Designer 7.90 software (PREMIER Biosoft International, Palo Alto, CA, USA) and are listed in Table S1. The qRT-PCR reaction was performed in 20 μL reactions containing 10 μL 2× Go Taq qPCR Master Mix (Promega, Madison, WI, USA). The PCR programs were 95°C for 2 min, 40 cycles at 95°C for 30 s, 60°C for
S. litura were reared in our laboratory at 25 ± 1°C, 14:10 h light/dark photoperiod, 65% ± 5% relative humidity on an artificial diet and sexed as pupae. RNA extraction and gene cloning The tissues were dissected from 3-day-old male and female moths and immediately transferred to Eppendorf tubes, frozen in liquid nitrogen and then stored at −80°C. Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Before reverse transcription, RNA was digested with DNase I. The cDNA template was synthesized with Oligo (dT)18 primers as anchor primers using Revert AidTM M-MuLV reverse transcriptase (Fermentas, Glen Burnie, MD, USA) at 42°C for 60 min. The reactions were stopped by heating for 5 min at 70°C. Gene-specific primers were designed to clone the SNMP fragments according to the two unigenes encoding putative SNMP1 (EZ982816) and SNMP2 (EZ982501) identified in Spodoptera littoralis, by Legeai et al. (2011). In order to obtain the full-length C
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1 min. The PCR products were then heated to 95°C for 15 s, cooled to 60°C for 1 min and heated again to 95°C for 15 s to measure the dissociation curves. In situ hybridization The histology was performed on tissues from 1- to 2day-old male moths. Antennae were isolated, embedded in Tissue-Tek freezing medium (R. Jung GmbH, Nussloch, Germany) and rapidly frozen at −22°C on the object holder. Cryosections (12 μm) of antennae were thaw mounted on slides and air dried at room temperature for at least 30 min. Subsequently, slides were treated at 4°C with 4% paraformaldehyde in 0.1 mol/L NaCO3 , pH 9.5 for 30 min,1× PBS (phosphate-buffered saline = 0.85% NaCl, 1.4 mmol/L KH2 PO4 , 8 mmol/L Na2 HPO4 , pH 7.1) for 1 min, 0.2 mol/L HCl for 10 min and PBS with 1% Triton X-100 for 2 min followed by two 30-s wash treatments in PBS. In situ hybridization with single antisense Digoxigenin (DIG)-labeled SlituSNMP1 RNA probes, as well as visualization was performed as described previously (Krieger et al., 2002). DIG-labeled and biotinlabeled antisense probes for S. litura were synthesized from linearized recombinant pGM-T vectors (TianGen, Beijing, China) containing the coding sequences of SlituSNMP1 and SlituSNMP2, using an SP6/T7 transcription kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. DIG-labeled probes were detected by the anti-DIG AP-conjugated antibody in combination with HNPP/Fast Red (Fluorescent Detection Set; Roche). For biotin-labeled probes the components and protocols of the TSA kit (Perkin Elmer, Waltham, MA, USA), including a strepavidin horseradish peroxidase-conjugate and fluorescein isothiocyanate-tyramides as substrates, were used. The sections were analyzed on both an Olympus microscope (Olympus, Tokyo, Japan) and LEICA DMIRE2 microscope (LEICA, Wetzlar, Germany). Results Identification of SlituSNMP genes According to the SNMP sequences identified from the expressed sequence tags of the S. littoralis antennae (Legeai et al., 2011), we designed specific primers to clone two cDNA fragments of SNMPs from S. litura antennae. The full-length cDNA of the two genes, SlituSNMP1 and SlituSNMP2, were obtained by RACE experiments. Sequence analysis showed that SlituSNMP1 gene was 1575 bp long encoding 524 amino acids, and SlituSNMP2 consisted of 1563 nucleotides that encoded 520 amino C 2014
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acids. The sequences of SlituSNMP1 and SlituSNMP2 have been deposited in GenBank with accession numbers KC571258 and KC571259, respectively. Online analysis showed that the theoretical pI of the two SlituSNMPs were 6.36 and 6.48, and the molecular weight of the two proteins were 59.08 kDa and 58.53 kDa, respectively. Transmembrane domain prediction with TMHMM showed that both protein sequences had two transmembrane regions with intracellular N- and C-terminal (Fig. 1).
Sequence comparison and phylogenetic analysis Amino acid sequences alignment with previously reported full-length SNMPs was made in insects including Lepidoptera, Coleoptera, Hymenoptera and Diptera, showed six conserved cysteine residues. Figure 2 only shows the alignment result from Lepidoptera SNMPs. Based on the alignment results, a phylogenetic tree was generated (Fig. 3), showing two distinct subgroups for SNMP1 and SNMP2. Sequence comparisons showed that the identities within the same SNMP subgroup were much higher than between SNMP subgroups. SltiuSNMP1 had the highest degree of identity with SexiSNMP1 (JX469106) at 93% and SlituSNMP2 was most identical with SexiSNMP2 (JX469107) at 92%. However, the identity between SlituSNMP1 and SlituSNMP2 was only 28%.
Tissue expression patterns of the SNMP genes RT-PCR experiments were carried out to determine the expression patterns of SlituSNMP genes. A housekeeping gene encoding actin was used to test the integrity of the cDNA templates. All PCR product bands matched the expected size. The results showed that SlituSNMP1 was predominantly expressed in male and female antennae, and also weakly expressed in male maxillary palps. In contrast, SlituSNMP2 were broadly expressed in olfactory tissues (antennae, proboscises, maxillary palps) and non-olfactory tissues (legs, genitalia) (Fig. 4A). qRTPCR was then conducted to further explore the expression level in different tissues. Expression of both SlituSNMP1 and SlituSNMP2 were most abundant in the antennae, especially for SlituSNMP1. Consistent with the RT-PCR, SlituSNMP2 could be slightly detected in other tissues (proboscises, maxillary palps, legs, genitalia). The relative expression levels of SlituSNMP1 and SlituSNMP2 transcripts in the female antennae were about 2-fold higher than the male antennae (Fig. 4B).
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Fig. 1 Nucleotide and deduced amino acid sequences of SlituSNMP1 (A) and SlituSNMP2 (B) from Spodoptera litura. The six cysteine resides conserved among all the identified SNMP sequences in insects are highlighted in red and boxed. The stop codons are marked with “-”. The black lines below the amino acid sequences indicate the two transmembrane domains.
In situ hybridization
hybridization signals were found at the base of short sensilla trichodea and long sensilla trichodea (Fig. 5B, D), with no signals in sensilla basiconica and sensilla chaetica (Fig. 5C, E). To visualize the relative expression patterns of SlituSNMP1 and SlituSNMP2 in antenna, DIG-labeled SlituSNMP1 probes and biotin-labeled SlituSNMP2 probes were combined to perform a double in situ hybridization on the same section, and the signals were visualized by different fluorescent colors.
To determine the cell expression patterns of the two SlituSNMPs, double in situ hybridization experiments were performed on cryosections of the male antenna. First, DIG-labeled SlituSNMP1-specific antisense probes were performed on longitudinal sections of the male antennae to visualize cells that express SlituSNMP1, with SlituSNMP1 sense strand probe as a control (Fig. 5A). The
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Fig. 2 Alignment of SNMP1 and SNMP2 from Lepidopteran insects. The six conserved cysteines are highlighted in red. Abbreviated species names and GenBank accession numbers of the amino acid sequences are listed in the Table S2.
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Fig. 3 Phylogenetic analysis of SNMPs in insects conducted in MEGA 5.0. C
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Fig. 4 Expression patterns of SlituSNMP1 and SlituSNMP2 genes in Spodoptera litura tissues. MA, male antenna; FA, female antenna; MP, male proboscises; FP, female proboscises; Mmp, male maxillary palps; Fmp, female maxillary palps; ML, male legs; FL, female legs; MG, male genitalia; FG, female genitalia
SlituSNMP1 and SlituSNMP2 are closely associated, but expressed in different cells (Fig. 6 and Fig. S1).
Discussion In this study, we identified two SNMPs, SlituSNMP1 and SlituSNMP2, from the moth S. litura. Amino acid sequence comparison shows that SlituSNMP1 and SlituSNMP2 have all the characteristics of SNMPs, especially two transmembrane domains, as well as six conserved cysteines suggested to form disulfide bridge in the large central extracellular loop (Rasmussen et al., 1998). Phylogenetic analysis indicates that the insect SNMPs, which are structurally different from odorant receptors (Krieger et al., 2002; Krieger et al., 2005; Nakagawa et al., 2005; Wang et al., 2011), are divided into two clear subgroups, SNMP1 and SNMP2, similar to previous reports (Nichols & Vogt, 2008; Vogt et al., 2009; Liu et al., 2013; Liu et al., 2014). Furthermore, in each of the two subgroups, the SNMPs from different Lepidoptera species cluster together, forming a clade distinct from SNMP homologues of Coleoptera, Diptera and Hymenoptera. This indicates potential for divergence of function across taxa during the evolutionary process. The expression pattern analysis showed that SlituSNMP1 was mainly expressed in male and female antenna, similar to previous reports in A. polyphemus (Rogers et al., C 2014
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1997), Amyelois transitella (Leal et al., 2009), Anopheles gambiae (Benton et al., 2007) and S. exigua (Liu et al., 2014). SNMP1 has been also reported to be widely distributed in different tissues in other insects such as Drosophila melangoster, Cnaphalocrocis medinalis and Agrotis ipsilon (Benton et al., 2007; Gu et al., 2013; Liu et al., 2013). SlituSNMP2 was expressed in both olfactory and non-olfactory tissues (legs and genitalia), similar to observations in D. melanogaster, Aedes aegypti, Cnaphalocrocis medinalis, Sesamia inferens, Agrotis ipsilon and S. exigua (Jin et al., 2008; Vogt et al., 2009; Liu et al., 2013; Gu et al., 2013; Zhang et al., 2013; Liu et al., 2014). This could be explained by the presence of chemosensory sensilla on these organs. The higher relative expression pattern of SlituSNMP1 and SlituSNMP2 in female antennae was reported in our study, which was just the opposite from previous studies (Liu et al., 2013; Gu et al., 2013). Rogers et al. (2001a) showed almost equal expression of MsexSNMP1 and MsexSNMP2 in the male and female antennae of Manduca sexta. qRT-PCR data in A. ipsilon revealed the expression of both SNMP1 and SNMP2 increased dramatically from 1 day before eclosion and reached the peak at the third day. This age-dependent expression profile was consistent with the pheromone production and mating behavior of A. ipsilon (Gu et al., 2013). The abundant expression of SlituSNMP1 in antenna indicates that it may be specifically involved in olfaction, while the
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Fig. 5 In situ hybridization of SlituSNMP1 in male antennae of Spodoptera litura. Digoxigenin (DIG)-labelled sense and antisense RNA probes were performed on longitudinal tissue sections of antennae. Signals were visualized using an anti-DIG antibody. (A) Sense probe as a control, (B) the signal in short sensilla trichodea, (C) sensilla basiconica not stained, (D) the signal in long sensilla trichodea, (E) sensilla chaetica not stained.
Fig. 6 Expression of SltiuSNMP1 and SlituSNMP2 in the antenna of Spodoptera litura. Digoxigenin-labeled SlituSNMP1 and biotinlabeled SlituSNMP2 probes were used in double in situ hybridization on a section through the male antenna. Cells expressing SlituSNMP1 and SlituSNMP2 are visualized by red (A) and green (B) fluorescence, respectively. The overlay of the transmitted-light channel with the red and green fluorescence channels is shown in (C).
broad expression patterns of the SlituSNMP2 suggest that it may function in not only olfaction organs, and may have different functions specific to the various tissues (Vogt et al., 2009). Benton et al. (2007) have demonstrated that DmelSNMP1 is required for the detection of the aggregation pheromone cVA and the activation of the moth
pheromone receptor, HvirPR13 ectopically expressed in the OR67d neuron. Jin et al. (2008) suggested that SNMP1 was required on the dendritic surface, where it was exposed to the sensillum lymph, supporting the idea that DmelSNMP1 functions directly in cVA signal transduction. These experiments imply that SNMP function may C
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be conserved across taxa and may function specifically in mediating pheromone detection and transduction through SNMP-OR interactions (Benton et al., 2007; Nichols & Vogt, 2008). The in situ hybridization results showed that SlituSNMP1 was expressed in only one olfactory neuron per sensillum, although there were generally two or three olfactory neurons in each sensillum. This observation is similar with the expression patterns of SNMP1 seen in A. polyphemus (Rogers et al., 1997). The expression of SlituSNMP1 in long sensilla trichodea indicates it may participate in pheromone reception, as these sensilla are mainly involved in pheromone detection (Krieger et al., 2009; Ljungberg et al., 1993; Gohl & Krieger, 2006; Grosse-Wilde et al., 2007; Liu et al., 2014). The doublelabeled signals showed that SlituSNMP1 and SlituSNMP2 are closely associated, but expressed in different cells. These different expression patterns in SNMP1 and SNMP2 suggest that there may be a functional differentiation between these genes or they could function similarly in different cell types. In another study, SexiSNMP1 and SexiSNMP2 were expressed in long and short trichoid as well as basiconic sensilla (Liu et al., 2014). There are two possibilities to address the distinct distribution pattern between the two related species: SNMPs may have different expression patterns among different species, or the difference in cutting angles during preparing the slices of antennae resulted in erroneous judgment of SNMP localization in sensilla. In sum, we identified two SNMP genes in S. litura and investigated the topographic expression in male antenna and the expression profile in different tissues in adults. This should be helpful for further functional research of SNMPs in this insect. Acknowledgments We would like to thank Li-Yan Yang for insect rearing, Shu-Wei Yan and Meng-Bo Guo for technical assistance in the qRT-PCR and in situ hybridization. This work was supported by the National Basic Research Program of China (Grant No. 2012CB114104), the National Natural Science Foundation of China (Grant No. 31230062 and 31071752) and Beijing Natural Science Foundation (6132028). References Benton, R., Vannice, K.S. and Vosshall, L.B. (2007) An essential role for a CD36-related receptor in pheromone detection in Drosophila. Nature, 450, 289–293. C 2014
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Febbraio, M. and Silverstein, R.L. (2007) CD36: implications in cardiovascular disease. International Journal of Biochemistry & Cell Biology, 39, 2012–2030. Forstner, M., Gohl, T., Gondesen, I., Raming, K., Breer, H. and Krieger, J. (2008) Differential expression of SNMP-1 and SNMP-2 proteins in pheromone-sensitive hairs of moths. Chemical Senses, 33, 291–299. Fukuwatari, T., Kawada, T., Tsuruta, M., Hiraoka, T., Iwanaga, T., Sugimoto, E. and Fushiki, T. (1997) Expression of the putative membrane fatty acid transporter (FAT) in taste buds of the circumvallate papillae in rats. FEBS Letters, 414, 461– 464. Gohl, T. and Krieger, J. (2006) Immunolocalization of a candidate pheromone receptor in the antenna of the male moth, Heliothis virescens. Invertebrate Neuroscience, 6, 13–21. Grosse-Wilde, E., Gohl, T., Bouche, E., Breer, H. and Krieger, J. (2007) Candidate pheromone receptors provide the basis for the response of distinct antennal neurons to pheromonal compounds. European Journal of Neuroscience, 25, 2364– 2373. Gu, S.H., Yang, R.N., Guo, M.B., Wang, G.R., Wu, K.M., Guo, Y.Y., Zhou, J.J. and Zhang, Y.J. (2013) Molecular identification and differential expression of sensory neuron membrane proteins in the antennae of the black cutworm moth Agrotis ipsilon. Journal of Insect Physiology, 59, 430–443. Jin, X., Ha, T.S. and Smith, D.P. (2008) SNMP is a signaling component required for pheromone sensitivity in Drosophila. Proceedings of the National Academy of Sciences of the United States of America, 105, 10996–11001. Krieger, J., Gondesen, I., Forstner, M., Gohl, T., Dewer, Y. and Breer, H. (2009) HR11 and HR13 receptor-expressing neurons are housed together in pheromone-responsive sensilla trichodea of male Heliothis virescens. Chemical Senses, 34, 469–477. Krieger, J., Grosse-Wilde, E., Gohl, T. and Breer, H. (2005) Candidate pheromone receptors of the silkmoth Bombyx mori. European Journal of Neuroscience, 21, 2167–2176. Krieger, J., Raming, K., Dewer, Y.M., Bette, S., Conzelmann, S. and Breer, H. (2002) A divergent gene family encoding candidate olfactory receptors of the moth Heliothis virescens. European Journal of Neuroscience, 16, 619–628. Leal, W.S., Ishida, Y., Pelletier, J., Xu, W., Rayo, J., Xu, X. and Ames, J.B. (2009) Olfactory proteins mediating chemical communication in the navel orangeworm moth, Amyelois transitella. PLoS ONE, 4: e7235. Legeai, F., Malpel, S., Montagne, N., Monsempes, C., Cousserans, F., Merlin, C., Francois, M.C., Maibeche-Coisne, M., Gavory, F., Poulain, J. and Jacquin-Joly, E. (2011) An expressed sequence tag collection from the male antennae of the noctuid moth Spodoptera littoralis: a resource for olfactory and pheromone detection research. BMC Genomics, 12, 86.
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Levy, E., Spahis, S., Sinnett, D., Peretti, N., Maupas-Schwalm, F., Delvin, E., Lambert, M. and Lavoie, M.A. (2007) Intestinal cholesterol transport proteins: an update and beyond. Current Opinion in Lipidology, 18, 310–318. Liu, C., Zhang, J., Liu, Y., Wang, G. and Dong, S. (2014) Expression of SNMP1 and SNMP2 genes in antennal sensilla of Spodoptera exigua (H¨ubner). Archives of Insect Biochemistry and Physiology, 85, 114–126. Liu, S., Zhang, Y.R., Zhou, W.W., Liang, Q.M., Yuan, X., Cheng, J., Zhu, Z.R. and Gong, Z.J. (2013) Identification and characterization of two sensory neuron membrane proteins from Cnaphalocrocis medinalis (Lepidoptera: Pyralidae). Archives of Insect Biochemistry and Physiology, 82, 29–42. Ljungberg, H., Anderson, P. and Hansson, B.S. (1993) Physiology and morphology of pheromone-specific sensilla on the antennae of male and female Spodoptera littoralis (Lepidoptera: Noctuidae). Journal of Insect Physiology, 39, 253– 260. Nakagawa, T., Sakurai, T., Nishioka, T. and Touhara, K. (2005) Insect sex-pheromone signals mediated by specific combinations of olfactory receptors. Science, 307, 1638–1642. Nassir, F., Wilson, B., Han, X., Gross, R.W. and Abumrad, N.A. (2007) CD36 is important for fatty acid and cholesterol uptake by the proximal but not distal intestine. Journal of Biological Chemistry, 282, 19493–19501. Nichols, Z. and Vogt, R.G. (2008) The SNMP/CD36 gene family in Diptera, Hymenoptera and Coleoptera: drosophila melanogaster, D. pseudoobscura, Anopheles gambiae, Aedes aegypti, Apis mellifera, and Tribolium castaneum. Insect Biochemistry and Molecular Biology, 38, 398–415. Rasmussen, J.T., Berglund, L., Rasmussen, M.S. and Petersen, T.E. (1998) Assignment of disulfide bridges in bovine CD36. European Journal of Biochemistry, 257, 488–494. Rogers, M.E., Krieger, J. and Vogt, R.G. (2001a) Antennal SNMPs (sensory neuron membrane proteins) of Lepidoptera define a unique family of invertebrate CD36-like proteins. Journal of Neurobiology, 49, 47–61.
Rogers, M.E., Steinbrecht, R.A. and Vogt, R.G. (2001b) Expression of SNMP-1 in olfactory neurons and sensilla of male and female antennae of the silkmoth Antheraea polyphemus. Cell & Tissue Research, 303, 433–446. Rogers, M.E., Sun, M., Lerner, M.R. and Vogt, R.G. (1997) Snmp-1, a novel membrane protein of olfactory neurons of the silk moth Antheraea polyphemus with homology to the CD36 family of membrane proteins. Journal of Biological Chemistry, 272, 14792–14799. Vogt, R.G., Miller, N.E., Litvack, R., Fandino, R.A., Sparks, J., Staples, J., Friedman, R. and Dickens, J.C. (2009) The insect SNMP gene family. Insect Biochemistry and Molecular Biology, 39, 448–456. Wang, G., Vasquez, G.M., Schal, C., Zwiebel, L.J. and Gould, F. (2011) Functional characterization of pheromone receptors in the tobacco budworm Heliothis virescens. Insect Molecular Biology, 20, 125–133. Zhang, Y.N., Jin, J.Y., Jin, R., Xia, Y.H., Zhou, J.J., Deng, J.Y. and Dong, S.L. (2013) Differential expression patterns in chemosensory and non-chemosensory tissues of putative chemosensory genes identified by transcriptome analysis of insect pest the purple stem borer Sesamia inferens (Walker). PLoS ONE, 8: e69715.
Accepted March 4, 2014
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Primers for gene cloning, RT-PCR, RACE PCR and end-to-end PCR. Table S2. Genbank accession number of the sequences used in phylogenetic analysis. Fig. S1. The control in the in situ hybridization.
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ORIGINAL ARTICLE
Identification and localization of two sensory neuron membrane proteins from Spodoptera litura (Lepidoptera: Noctuidae) Jin Zhang1,2 , Yang Liu1 , William B. Walker3 , Shuang-Lin Dong2 and Gui-Rong Wang1 1 State
Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural
Sciences, Beijing, 2 Education Ministry Key Laboratory of Integrated Management of Crop Disease and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing, China and 3 Swedish University of Agricultural Sciences, Department of Plant Protection Biology, Chemical Ecology Research Group, Alnarp, Sweden
Abstract Sensory neuron membrane proteins (SNMPs), which are located on the dendritic membrane of olfactory sensory neurons (OSNs), are proposed to be associated with odor reception in insects. Recent studies have demonstrated that SNMP1 is essential for electrophysiological responses of OSNs to the sex pheromone, cis-vaccenyl acetate (cVA) in Drosophila melanogaster. To investigate the function of Lepidoptera SNMPs, we cloned two SNMP genes, SlituSNMP1 and SltiuSNMP2, from Spodoptera litura (Lepidoptera: Noctuidae). Sequence alignment and phylogenetic analysis showed that both genes bear the general characteristics of SNMPs, including six conserved cysteine residues and two transmembrane domains. Further expression profile experiments showed that SlituSNMP1 is mainly expressed in the antenna, while SlituSNMP2 is broadly expressed in various tissues. By in situ hybridization experiments, it was found that SlituSNMP1 expressing cells are surrounded by the SlituSNMP2 expressing cells in the pheromone sensitive sensilla, suggesting different functions of the two SNMPs in insect olfaction. Key words In situ hybridization, Spodoptera litura, sensory neuron membrane proteins (SNMPs)
Introduction Sensory neuron membrane proteins (SNMPs), first identified in Lepidoptera insects, are transmembrane proteins localized in dendritic membrane of the pheromone-sensing olfactory receptor neurons (ORNs) and are proposed to play an important role in pheromone reception in insects (Rogers et al., 1997; Rogers et al., 2001b; Benton et al., 2007; Forstner et al., 2008; Jin et al., 2008). SNMPs belong to a gene family of the human fatty
Correspondence: Gui-Rong Wang, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China. Tel: +86 010 62816947, Fax: +86 010 62896114; Email: [email protected]
acid transporter (FAT), CD36, which is characterized by two transmembrane domains and is mainly involved in the recognition of fatty acids, cholesterol and proteinaceous compounds in cells (Rogers et al., 1997; Rasmussen et al., 1998; Levy et al., 2007; Nassir et al., 2007; Febbraio & Silverstein, 2007; Fukuwatari et al., 1997). In the last decade, two SNMP orthologues (SNMP1 and SNMP2) have been characterized in several insect orders, including Lepidoptera, Hymenoptera, Diptera, Coleoptera and so on (Nichols & Vogt, 2008; Rogers et al., 2001a; Vogt et al., 2009; Forstner et al., 2008). Studies on Drosophila melanogaster demonstrated that DmelSNMP1 is essential in detecting the fruit fly pheromone cis-vaccenyl acetate (cVA) (Benton et al., 2007; Jin et al., 2008). Additionally, DmelSNMP1 was also required for the activation of the ectopically expressed pheromone receptor HvirOR13 (Heliothis 1
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virescens) by (Z)-11-hexadecenal in OR67d-expressing neurons (Benton et al., 2007). Forstner et al. (2008) reported that HvirSNMP1 is coexpressed with HvirOR13 in the same ORNs in the antenna, but HvirSNMP2 is expressed in the supporting cells that surround the HvirOR13 neurons. A similar expression pattern was also found for ApolSNMP1 and ApolSNMP2 on the antennae of male Antheraea polyphemus (Forstner et al., 2008). Recently the SNMPs in Cnaphalocrocis medinalis, Agrotis ipsilon and S. exigua have been identified and discussed (Liu et al., 2013; Gu et al., 2013; Liu et al., 2014). The exact function of SNMPs in moths is still unknown, but the essential role in pheromone reception in D. melanogaster and the broad expression in pheromone sensing sensilla indicate they may be involved in pheromone detection in Lepidoptera. To elucidate whether the SNMP expression patterns are identical in Lepidoptera or distinct in some species, we identified two SNMPs (SlituSNMP1 and SlituSNMP2) from the common cutworm, Spodoptera litura, and investigated the expression patterns of the two genes regarding different tissues and genders, and finally, determined the topographic and cell-specific expression of the two SNMPs in male antennae.
open reading frame sequences, rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) was performed using SMARTerTM RACE cDNA amplification kit (Clontech, Mountain View, CA, USA) for 5’ and 3’ ends amplification. The full-length sequences were assembled based on reverse transcription PCR (RT-PCR) and RACE results and then confirmed by end-to-end PCR using specific primers designed at both ends. The primers are listed in Table S1. Bioinformatic analysis Homologous SNMP sequences were searched from GenBank. Transmembrane domains of SlituSNMPs were predicted with TMHMM (http://www. cbs.dtu.dk/services/TMHMM). The unrooted consensus neighbor-joining (NJ) tree was constructed using the NJ method and evaluated by the bootstrap procedure based on 1000 replicates with pairwise gap deletions in MEGA 5.0 software (The Biodesign Institute, Center for Evolutionary Functional Genomics, Tempe, AZ, USA). The theoretical isoelectric points (pI) and molecular weights of the deduced SlituSNMP1 and SlituSNMP2 were obtained by using ExPASy server software (http://www.expasy.org/tools/protparam.html).
Materials and methods Tissue expression pattern
Insects
To confirm the expression profiles of SlituSNMP1 and SlituSNMP2, antennae, proboscises, maxillary palps, legs and genitalia were isolated from male and female adults of S. litura. RNA extraction and cDNA synthesis were performed as described above. A “house-keeping” gene of S. litura encoding actin (DQ494753.1) was used to verify the integrity of the cDNA. The PCR programs were 95°C for 3 min, 35 cycles at 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s, with a final 10 min incubation at 72°C. PCR products were analyzed on 2.0% agarose gels. Quantitative RT-PCR (qRT-PCR) analysis was conducted on the ABI Prism 7500 Fast Detection System (Applied Biosystems, Carlsbad, CA, USA). Two reference genes GAPDH (GenBank No. HQ012003.2) and elongation factor 1-a (GenBank No. KC007373.1) were used to normalize the target gene expression and to correct sample-tosample variation. The qRT-PCR primers were designed using the Beacon Designer 7.90 software (PREMIER Biosoft International, Palo Alto, CA, USA) and are listed in Table S1. The qRT-PCR reaction was performed in 20 μL reactions containing 10 μL 2× Go Taq qPCR Master Mix (Promega, Madison, WI, USA). The PCR programs were 95°C for 2 min, 40 cycles at 95°C for 30 s, 60°C for
S. litura were reared in our laboratory at 25 ± 1°C, 14:10 h light/dark photoperiod, 65% ± 5% relative humidity on an artificial diet and sexed as pupae. RNA extraction and gene cloning The tissues were dissected from 3-day-old male and female moths and immediately transferred to Eppendorf tubes, frozen in liquid nitrogen and then stored at −80°C. Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Before reverse transcription, RNA was digested with DNase I. The cDNA template was synthesized with Oligo (dT)18 primers as anchor primers using Revert AidTM M-MuLV reverse transcriptase (Fermentas, Glen Burnie, MD, USA) at 42°C for 60 min. The reactions were stopped by heating for 5 min at 70°C. Gene-specific primers were designed to clone the SNMP fragments according to the two unigenes encoding putative SNMP1 (EZ982816) and SNMP2 (EZ982501) identified in Spodoptera littoralis, by Legeai et al. (2011). In order to obtain the full-length C
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1 min. The PCR products were then heated to 95°C for 15 s, cooled to 60°C for 1 min and heated again to 95°C for 15 s to measure the dissociation curves. In situ hybridization The histology was performed on tissues from 1- to 2day-old male moths. Antennae were isolated, embedded in Tissue-Tek freezing medium (R. Jung GmbH, Nussloch, Germany) and rapidly frozen at −22°C on the object holder. Cryosections (12 μm) of antennae were thaw mounted on slides and air dried at room temperature for at least 30 min. Subsequently, slides were treated at 4°C with 4% paraformaldehyde in 0.1 mol/L NaCO3 , pH 9.5 for 30 min,1× PBS (phosphate-buffered saline = 0.85% NaCl, 1.4 mmol/L KH2 PO4 , 8 mmol/L Na2 HPO4 , pH 7.1) for 1 min, 0.2 mol/L HCl for 10 min and PBS with 1% Triton X-100 for 2 min followed by two 30-s wash treatments in PBS. In situ hybridization with single antisense Digoxigenin (DIG)-labeled SlituSNMP1 RNA probes, as well as visualization was performed as described previously (Krieger et al., 2002). DIG-labeled and biotinlabeled antisense probes for S. litura were synthesized from linearized recombinant pGM-T vectors (TianGen, Beijing, China) containing the coding sequences of SlituSNMP1 and SlituSNMP2, using an SP6/T7 transcription kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. DIG-labeled probes were detected by the anti-DIG AP-conjugated antibody in combination with HNPP/Fast Red (Fluorescent Detection Set; Roche). For biotin-labeled probes the components and protocols of the TSA kit (Perkin Elmer, Waltham, MA, USA), including a strepavidin horseradish peroxidase-conjugate and fluorescein isothiocyanate-tyramides as substrates, were used. The sections were analyzed on both an Olympus microscope (Olympus, Tokyo, Japan) and LEICA DMIRE2 microscope (LEICA, Wetzlar, Germany). Results Identification of SlituSNMP genes According to the SNMP sequences identified from the expressed sequence tags of the S. littoralis antennae (Legeai et al., 2011), we designed specific primers to clone two cDNA fragments of SNMPs from S. litura antennae. The full-length cDNA of the two genes, SlituSNMP1 and SlituSNMP2, were obtained by RACE experiments. Sequence analysis showed that SlituSNMP1 gene was 1575 bp long encoding 524 amino acids, and SlituSNMP2 consisted of 1563 nucleotides that encoded 520 amino C 2014
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acids. The sequences of SlituSNMP1 and SlituSNMP2 have been deposited in GenBank with accession numbers KC571258 and KC571259, respectively. Online analysis showed that the theoretical pI of the two SlituSNMPs were 6.36 and 6.48, and the molecular weight of the two proteins were 59.08 kDa and 58.53 kDa, respectively. Transmembrane domain prediction with TMHMM showed that both protein sequences had two transmembrane regions with intracellular N- and C-terminal (Fig. 1).
Sequence comparison and phylogenetic analysis Amino acid sequences alignment with previously reported full-length SNMPs was made in insects including Lepidoptera, Coleoptera, Hymenoptera and Diptera, showed six conserved cysteine residues. Figure 2 only shows the alignment result from Lepidoptera SNMPs. Based on the alignment results, a phylogenetic tree was generated (Fig. 3), showing two distinct subgroups for SNMP1 and SNMP2. Sequence comparisons showed that the identities within the same SNMP subgroup were much higher than between SNMP subgroups. SltiuSNMP1 had the highest degree of identity with SexiSNMP1 (JX469106) at 93% and SlituSNMP2 was most identical with SexiSNMP2 (JX469107) at 92%. However, the identity between SlituSNMP1 and SlituSNMP2 was only 28%.
Tissue expression patterns of the SNMP genes RT-PCR experiments were carried out to determine the expression patterns of SlituSNMP genes. A housekeeping gene encoding actin was used to test the integrity of the cDNA templates. All PCR product bands matched the expected size. The results showed that SlituSNMP1 was predominantly expressed in male and female antennae, and also weakly expressed in male maxillary palps. In contrast, SlituSNMP2 were broadly expressed in olfactory tissues (antennae, proboscises, maxillary palps) and non-olfactory tissues (legs, genitalia) (Fig. 4A). qRTPCR was then conducted to further explore the expression level in different tissues. Expression of both SlituSNMP1 and SlituSNMP2 were most abundant in the antennae, especially for SlituSNMP1. Consistent with the RT-PCR, SlituSNMP2 could be slightly detected in other tissues (proboscises, maxillary palps, legs, genitalia). The relative expression levels of SlituSNMP1 and SlituSNMP2 transcripts in the female antennae were about 2-fold higher than the male antennae (Fig. 4B).
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Fig. 1 Nucleotide and deduced amino acid sequences of SlituSNMP1 (A) and SlituSNMP2 (B) from Spodoptera litura. The six cysteine resides conserved among all the identified SNMP sequences in insects are highlighted in red and boxed. The stop codons are marked with “-”. The black lines below the amino acid sequences indicate the two transmembrane domains.
In situ hybridization
hybridization signals were found at the base of short sensilla trichodea and long sensilla trichodea (Fig. 5B, D), with no signals in sensilla basiconica and sensilla chaetica (Fig. 5C, E). To visualize the relative expression patterns of SlituSNMP1 and SlituSNMP2 in antenna, DIG-labeled SlituSNMP1 probes and biotin-labeled SlituSNMP2 probes were combined to perform a double in situ hybridization on the same section, and the signals were visualized by different fluorescent colors.
To determine the cell expression patterns of the two SlituSNMPs, double in situ hybridization experiments were performed on cryosections of the male antenna. First, DIG-labeled SlituSNMP1-specific antisense probes were performed on longitudinal sections of the male antennae to visualize cells that express SlituSNMP1, with SlituSNMP1 sense strand probe as a control (Fig. 5A). The
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Fig. 2 Alignment of SNMP1 and SNMP2 from Lepidopteran insects. The six conserved cysteines are highlighted in red. Abbreviated species names and GenBank accession numbers of the amino acid sequences are listed in the Table S2.
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Fig. 3 Phylogenetic analysis of SNMPs in insects conducted in MEGA 5.0. C
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Fig. 4 Expression patterns of SlituSNMP1 and SlituSNMP2 genes in Spodoptera litura tissues. MA, male antenna; FA, female antenna; MP, male proboscises; FP, female proboscises; Mmp, male maxillary palps; Fmp, female maxillary palps; ML, male legs; FL, female legs; MG, male genitalia; FG, female genitalia
SlituSNMP1 and SlituSNMP2 are closely associated, but expressed in different cells (Fig. 6 and Fig. S1).
Discussion In this study, we identified two SNMPs, SlituSNMP1 and SlituSNMP2, from the moth S. litura. Amino acid sequence comparison shows that SlituSNMP1 and SlituSNMP2 have all the characteristics of SNMPs, especially two transmembrane domains, as well as six conserved cysteines suggested to form disulfide bridge in the large central extracellular loop (Rasmussen et al., 1998). Phylogenetic analysis indicates that the insect SNMPs, which are structurally different from odorant receptors (Krieger et al., 2002; Krieger et al., 2005; Nakagawa et al., 2005; Wang et al., 2011), are divided into two clear subgroups, SNMP1 and SNMP2, similar to previous reports (Nichols & Vogt, 2008; Vogt et al., 2009; Liu et al., 2013; Liu et al., 2014). Furthermore, in each of the two subgroups, the SNMPs from different Lepidoptera species cluster together, forming a clade distinct from SNMP homologues of Coleoptera, Diptera and Hymenoptera. This indicates potential for divergence of function across taxa during the evolutionary process. The expression pattern analysis showed that SlituSNMP1 was mainly expressed in male and female antenna, similar to previous reports in A. polyphemus (Rogers et al., C 2014
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1997), Amyelois transitella (Leal et al., 2009), Anopheles gambiae (Benton et al., 2007) and S. exigua (Liu et al., 2014). SNMP1 has been also reported to be widely distributed in different tissues in other insects such as Drosophila melangoster, Cnaphalocrocis medinalis and Agrotis ipsilon (Benton et al., 2007; Gu et al., 2013; Liu et al., 2013). SlituSNMP2 was expressed in both olfactory and non-olfactory tissues (legs and genitalia), similar to observations in D. melanogaster, Aedes aegypti, Cnaphalocrocis medinalis, Sesamia inferens, Agrotis ipsilon and S. exigua (Jin et al., 2008; Vogt et al., 2009; Liu et al., 2013; Gu et al., 2013; Zhang et al., 2013; Liu et al., 2014). This could be explained by the presence of chemosensory sensilla on these organs. The higher relative expression pattern of SlituSNMP1 and SlituSNMP2 in female antennae was reported in our study, which was just the opposite from previous studies (Liu et al., 2013; Gu et al., 2013). Rogers et al. (2001a) showed almost equal expression of MsexSNMP1 and MsexSNMP2 in the male and female antennae of Manduca sexta. qRT-PCR data in A. ipsilon revealed the expression of both SNMP1 and SNMP2 increased dramatically from 1 day before eclosion and reached the peak at the third day. This age-dependent expression profile was consistent with the pheromone production and mating behavior of A. ipsilon (Gu et al., 2013). The abundant expression of SlituSNMP1 in antenna indicates that it may be specifically involved in olfaction, while the
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Fig. 5 In situ hybridization of SlituSNMP1 in male antennae of Spodoptera litura. Digoxigenin (DIG)-labelled sense and antisense RNA probes were performed on longitudinal tissue sections of antennae. Signals were visualized using an anti-DIG antibody. (A) Sense probe as a control, (B) the signal in short sensilla trichodea, (C) sensilla basiconica not stained, (D) the signal in long sensilla trichodea, (E) sensilla chaetica not stained.
Fig. 6 Expression of SltiuSNMP1 and SlituSNMP2 in the antenna of Spodoptera litura. Digoxigenin-labeled SlituSNMP1 and biotinlabeled SlituSNMP2 probes were used in double in situ hybridization on a section through the male antenna. Cells expressing SlituSNMP1 and SlituSNMP2 are visualized by red (A) and green (B) fluorescence, respectively. The overlay of the transmitted-light channel with the red and green fluorescence channels is shown in (C).
broad expression patterns of the SlituSNMP2 suggest that it may function in not only olfaction organs, and may have different functions specific to the various tissues (Vogt et al., 2009). Benton et al. (2007) have demonstrated that DmelSNMP1 is required for the detection of the aggregation pheromone cVA and the activation of the moth
pheromone receptor, HvirPR13 ectopically expressed in the OR67d neuron. Jin et al. (2008) suggested that SNMP1 was required on the dendritic surface, where it was exposed to the sensillum lymph, supporting the idea that DmelSNMP1 functions directly in cVA signal transduction. These experiments imply that SNMP function may C
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be conserved across taxa and may function specifically in mediating pheromone detection and transduction through SNMP-OR interactions (Benton et al., 2007; Nichols & Vogt, 2008). The in situ hybridization results showed that SlituSNMP1 was expressed in only one olfactory neuron per sensillum, although there were generally two or three olfactory neurons in each sensillum. This observation is similar with the expression patterns of SNMP1 seen in A. polyphemus (Rogers et al., 1997). The expression of SlituSNMP1 in long sensilla trichodea indicates it may participate in pheromone reception, as these sensilla are mainly involved in pheromone detection (Krieger et al., 2009; Ljungberg et al., 1993; Gohl & Krieger, 2006; Grosse-Wilde et al., 2007; Liu et al., 2014). The doublelabeled signals showed that SlituSNMP1 and SlituSNMP2 are closely associated, but expressed in different cells. These different expression patterns in SNMP1 and SNMP2 suggest that there may be a functional differentiation between these genes or they could function similarly in different cell types. In another study, SexiSNMP1 and SexiSNMP2 were expressed in long and short trichoid as well as basiconic sensilla (Liu et al., 2014). There are two possibilities to address the distinct distribution pattern between the two related species: SNMPs may have different expression patterns among different species, or the difference in cutting angles during preparing the slices of antennae resulted in erroneous judgment of SNMP localization in sensilla. In sum, we identified two SNMP genes in S. litura and investigated the topographic expression in male antenna and the expression profile in different tissues in adults. This should be helpful for further functional research of SNMPs in this insect. Acknowledgments We would like to thank Li-Yan Yang for insect rearing, Shu-Wei Yan and Meng-Bo Guo for technical assistance in the qRT-PCR and in situ hybridization. This work was supported by the National Basic Research Program of China (Grant No. 2012CB114104), the National Natural Science Foundation of China (Grant No. 31230062 and 31071752) and Beijing Natural Science Foundation (6132028). References Benton, R., Vannice, K.S. and Vosshall, L.B. (2007) An essential role for a CD36-related receptor in pheromone detection in Drosophila. Nature, 450, 289–293. C 2014
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Febbraio, M. and Silverstein, R.L. (2007) CD36: implications in cardiovascular disease. International Journal of Biochemistry & Cell Biology, 39, 2012–2030. Forstner, M., Gohl, T., Gondesen, I., Raming, K., Breer, H. and Krieger, J. (2008) Differential expression of SNMP-1 and SNMP-2 proteins in pheromone-sensitive hairs of moths. Chemical Senses, 33, 291–299. Fukuwatari, T., Kawada, T., Tsuruta, M., Hiraoka, T., Iwanaga, T., Sugimoto, E. and Fushiki, T. (1997) Expression of the putative membrane fatty acid transporter (FAT) in taste buds of the circumvallate papillae in rats. FEBS Letters, 414, 461– 464. Gohl, T. and Krieger, J. (2006) Immunolocalization of a candidate pheromone receptor in the antenna of the male moth, Heliothis virescens. Invertebrate Neuroscience, 6, 13–21. Grosse-Wilde, E., Gohl, T., Bouche, E., Breer, H. and Krieger, J. (2007) Candidate pheromone receptors provide the basis for the response of distinct antennal neurons to pheromonal compounds. European Journal of Neuroscience, 25, 2364– 2373. Gu, S.H., Yang, R.N., Guo, M.B., Wang, G.R., Wu, K.M., Guo, Y.Y., Zhou, J.J. and Zhang, Y.J. (2013) Molecular identification and differential expression of sensory neuron membrane proteins in the antennae of the black cutworm moth Agrotis ipsilon. Journal of Insect Physiology, 59, 430–443. Jin, X., Ha, T.S. and Smith, D.P. (2008) SNMP is a signaling component required for pheromone sensitivity in Drosophila. Proceedings of the National Academy of Sciences of the United States of America, 105, 10996–11001. Krieger, J., Gondesen, I., Forstner, M., Gohl, T., Dewer, Y. and Breer, H. (2009) HR11 and HR13 receptor-expressing neurons are housed together in pheromone-responsive sensilla trichodea of male Heliothis virescens. Chemical Senses, 34, 469–477. Krieger, J., Grosse-Wilde, E., Gohl, T. and Breer, H. (2005) Candidate pheromone receptors of the silkmoth Bombyx mori. European Journal of Neuroscience, 21, 2167–2176. Krieger, J., Raming, K., Dewer, Y.M., Bette, S., Conzelmann, S. and Breer, H. (2002) A divergent gene family encoding candidate olfactory receptors of the moth Heliothis virescens. European Journal of Neuroscience, 16, 619–628. Leal, W.S., Ishida, Y., Pelletier, J., Xu, W., Rayo, J., Xu, X. and Ames, J.B. (2009) Olfactory proteins mediating chemical communication in the navel orangeworm moth, Amyelois transitella. PLoS ONE, 4: e7235. Legeai, F., Malpel, S., Montagne, N., Monsempes, C., Cousserans, F., Merlin, C., Francois, M.C., Maibeche-Coisne, M., Gavory, F., Poulain, J. and Jacquin-Joly, E. (2011) An expressed sequence tag collection from the male antennae of the noctuid moth Spodoptera littoralis: a resource for olfactory and pheromone detection research. BMC Genomics, 12, 86.
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Levy, E., Spahis, S., Sinnett, D., Peretti, N., Maupas-Schwalm, F., Delvin, E., Lambert, M. and Lavoie, M.A. (2007) Intestinal cholesterol transport proteins: an update and beyond. Current Opinion in Lipidology, 18, 310–318. Liu, C., Zhang, J., Liu, Y., Wang, G. and Dong, S. (2014) Expression of SNMP1 and SNMP2 genes in antennal sensilla of Spodoptera exigua (H¨ubner). Archives of Insect Biochemistry and Physiology, 85, 114–126. Liu, S., Zhang, Y.R., Zhou, W.W., Liang, Q.M., Yuan, X., Cheng, J., Zhu, Z.R. and Gong, Z.J. (2013) Identification and characterization of two sensory neuron membrane proteins from Cnaphalocrocis medinalis (Lepidoptera: Pyralidae). Archives of Insect Biochemistry and Physiology, 82, 29–42. Ljungberg, H., Anderson, P. and Hansson, B.S. (1993) Physiology and morphology of pheromone-specific sensilla on the antennae of male and female Spodoptera littoralis (Lepidoptera: Noctuidae). Journal of Insect Physiology, 39, 253– 260. Nakagawa, T., Sakurai, T., Nishioka, T. and Touhara, K. (2005) Insect sex-pheromone signals mediated by specific combinations of olfactory receptors. Science, 307, 1638–1642. Nassir, F., Wilson, B., Han, X., Gross, R.W. and Abumrad, N.A. (2007) CD36 is important for fatty acid and cholesterol uptake by the proximal but not distal intestine. Journal of Biological Chemistry, 282, 19493–19501. Nichols, Z. and Vogt, R.G. (2008) The SNMP/CD36 gene family in Diptera, Hymenoptera and Coleoptera: drosophila melanogaster, D. pseudoobscura, Anopheles gambiae, Aedes aegypti, Apis mellifera, and Tribolium castaneum. Insect Biochemistry and Molecular Biology, 38, 398–415. Rasmussen, J.T., Berglund, L., Rasmussen, M.S. and Petersen, T.E. (1998) Assignment of disulfide bridges in bovine CD36. European Journal of Biochemistry, 257, 488–494. Rogers, M.E., Krieger, J. and Vogt, R.G. (2001a) Antennal SNMPs (sensory neuron membrane proteins) of Lepidoptera define a unique family of invertebrate CD36-like proteins. Journal of Neurobiology, 49, 47–61.
Rogers, M.E., Steinbrecht, R.A. and Vogt, R.G. (2001b) Expression of SNMP-1 in olfactory neurons and sensilla of male and female antennae of the silkmoth Antheraea polyphemus. Cell & Tissue Research, 303, 433–446. Rogers, M.E., Sun, M., Lerner, M.R. and Vogt, R.G. (1997) Snmp-1, a novel membrane protein of olfactory neurons of the silk moth Antheraea polyphemus with homology to the CD36 family of membrane proteins. Journal of Biological Chemistry, 272, 14792–14799. Vogt, R.G., Miller, N.E., Litvack, R., Fandino, R.A., Sparks, J., Staples, J., Friedman, R. and Dickens, J.C. (2009) The insect SNMP gene family. Insect Biochemistry and Molecular Biology, 39, 448–456. Wang, G., Vasquez, G.M., Schal, C., Zwiebel, L.J. and Gould, F. (2011) Functional characterization of pheromone receptors in the tobacco budworm Heliothis virescens. Insect Molecular Biology, 20, 125–133. Zhang, Y.N., Jin, J.Y., Jin, R., Xia, Y.H., Zhou, J.J., Deng, J.Y. and Dong, S.L. (2013) Differential expression patterns in chemosensory and non-chemosensory tissues of putative chemosensory genes identified by transcriptome analysis of insect pest the purple stem borer Sesamia inferens (Walker). PLoS ONE, 8: e69715.
Accepted March 4, 2014
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Primers for gene cloning, RT-PCR, RACE PCR and end-to-end PCR. Table S2. Genbank accession number of the sequences used in phylogenetic analysis. Fig. S1. The control in the in situ hybridization.
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