J Chem Ecol DOI 10.1007/s10886-015-0574-x

Expression Analysis and Binding Assays in the Chemosensory Protein Gene Family Indicate Multiple Roles in Helicoverpa armigera Zhao-Qun Li 1,2 & Shuai Zhang 1 & Jun-Yu Luo 1 & Jing Zhu 1 & Jin-Jie Cui 1 & Shuang-Lin Dong 2

Received: 19 October 2014 / Revised: 4 February 2015 / Accepted: 6 March 2015 # Springer Science+Business Media New York 2015

Abstract Chemosensory proteins (CSPs) have been proposed to capture and transport hydrophobic chemicals to receptors on sensory neurons. We identified and cloned 24 CSP genes to better understand the physiological function of CSPs in Helicoverpa armigera. Quantitative real-time polymerase chain reaction assays indicate that CSP genes are ubiquitously expressed in adult H. armigera tissues. Broad expression patterns in adult tissues suggest that CSPs are involved in a diverse range of cellular processes, including chemosensation as well as other functions not related to chemosensation. The H. armigera CSPs that were highly transcribed in sensory organs or pheromone glands (HarmCSPs 6, 9, 18, 19), were recombinantly expressed in bacteria to explore their function. Fluorescent competitive binding assays were used to measure the binding affinities of these CSPs against 85 plant volatiles and 4 pheromone components. HarmCSP6 displays high binding affinity for pheromone components, whereas the other three proteins do not show affinities for any of the com-

Electronic supplementary material The online version of this article (doi:10.1007/s10886-015-0574-x) contains supplementary material, which is available to authorized users. * Jin-Jie Cui [email protected] Shuang-Lin Dong [email protected] 1

State Key Laboratory of Cotton Biology, Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang, Henan, China

2

Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing, China

pounds tested. HarmCSP6 is expressed in numerous cells located in or close to long sensilla trichodea on the antennae of both males and females. These results suggest that HarmCSP6 may be involved in transporting female sex pheromones in H. armigera. Keywords Helicoverpa armigera . Chemosensory proteins . Fluorescence binding assay . In Situ hybridization

Abbreviations CSP Chemosensory protein OBP odorant-binding protein OS-D olfactory specific protein-D qRT-PCR quantitative real-time polymerase chain reaction Nr Non-redundant protein sequences database 1-NPN 1-N-phenylnaphthylamine Ki Dissociation constant PBP Pheromone binding protein

Introduction A sophisticated olfactory system is crucial for the survival and reproduction of many insects, particularly as it affects behaviors that include feeding, mating, toxin avoidance, and negative taxis (Zhou 2010). Insect olfaction is mediated by specific olfactory sensory neurons located in the sensilla of chemosensory organs, which abound with small soluble binding proteins (Leal 2012). These small soluble proteins are of two major types: Odorant-Binding Proteins (OBPs) and Chemosensory Proteins (CSPs) (Pelosi et al. 2005; Pikielny et al. 1994). Odorant-Binding Proteins, which occur in high

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concentrations at olfactory sensilla, generally are thought to bind, transport, and deliver exogenous odorant molecules to odorant receptors on dendrite membranes (Leal 2012; Vogt 2003, 2004; Zhou 2010). Chemosensory Proteins, however, are a poorly understood component of the peripheral insect chemosensory system. The first identified member of the CSP family, named Olfactory Specific protein D (OS-D), was purified from the antennae of Drosophila melanogaster (McDonald and Rosbash 2001; Pikielny et al. 1994). These proteins were known originally as Sensory Appendage Proteins (Robertson et al. 1999), before being named CSPs (Angeli et al. 1999). Thereafter, many CSPs were isolated and cloned from several insect orders, including Lepidoptera (Gong et al. 2007), Orthoptera (Ban et al. 2003), Hymenoptera (Forêt et al. 2007), Hemiptera (Zhou et al. 2010), and Neuroptera (Li et al. 2013b). Compared with OBPs, which are usually specifically expressed in antennae, CSPs are broadly expressed in many organs, including antennae (González et al. 2009; JacquinJoly et al. 2001), proboscises (Nagnan-Le Meillour et al. 2000), legs (Kitabayashi et al. 1998), wings (Ban et al. 2003), and pheromone glands (Jacquin-Joly et al. 2001), as well as other tissues. This broad and diverse expression pattern suggests that CSPs may play multiple roles, beyond chemosensation. CSPs highly expressed in antenna have suggested chemosensory functions in Hymenoptera and Lepidoptera (González et al. 2009; Qiao et al. 2013; Zhang et al. 2014). Other CSPs abundant in antennae have been implicated as serving roles in the behavioral phase shift from gregarious to solitary (Guo et al. 2011). In Spodoptera exigua, CSP3 has been implicated in ovipositioning and egg hatching (Gong et al. 2012), while in Periplaneta americana CSP10 seems to be a major extracellular matrix protein during limb regeneration (Kitabayashi et al. 1998). Several studies indicate CSPs may be involved in immune response (Oduol et al. 2000). CSPs are, therefore, likely to perform many diverse tasks from behavior to various physiological processes. The study of chemosensation in the cotton bollworm, Helicoverpa armigera, has practical applications through the potential development of new pest-control strategies. Here, we identified 24 CSP genes from H. armigera. Furthermore, tissue expression profiles of these 24 genes were determined by using quantitative real-time polymerase chain reaction (qRT-PCR) assays. The results indicate that H. armigera CSPs (HarmCSPs) are ubiquitously expressed in adult tissues. Four CSPs were expressed using a bacterial expression system, and a competitive fluorescence binding assay showed that the HarmCSP6 protein has high binding affinities to pheromone components. In situ hybridization revealed that HarmCSP6 are expressed at the base of long and short sensilla trichodea in the antennae of both males and females.

Methods and Materials Insects Rearing and Tissue Collection Helicoverpa armigera was reared at 25±1 °C, with a 14:10 h L:D photoperiod, at 65±5 % relative humidity in the laboratory on an artificial diet, mainly consisting of soybean meal, maize flour, and wheat germ. Pupae were sexed and kept separately in cages for emergence. Upon eclosion, adults were provided with a 10 % honey solution. Antennae, heads (without antennae and proboscises), thoraxes, abdomen (without pheromone glands), legs, wings, and pheromone glands of 3-d-old virgin adults were collected separately to determine CSP gene tissue expression profiles. All collected tissues were transferred to Eppendorf tubes, immediately froze in liquid nitrogen, and stored at −80 °C until use. RNA Isolation and cDNA Synthesis Total RNAwas extracted using a SV Total Isolation System (Promega, Medison, WI, USA) following the manufacturer’s protocol. RNA quality was checked by spectrophotometer (NanoDrop 2000c, Thermo Fisher Scientific, USA). cDNA templates were synthesized using a Reverse Transcription System (Promega) according to the user manual. Sequence Alignment and Phylogenetic Analysis Potential CSP gene cDNAs were isolated from our cDNA library, and searched against the Non-redundant protein sequences database (Nr) using BLASTX with default parameters (http:// www.ncbi.nlm.nih.gov/). Twenty four CSP positive sequences were verified using RT-PCR and sequencing. An amino acid sequence alignment of the corresponding CSPs was created using ClustalX 2.0 (Larkin et al. 2007), and hand edited and visualized using Jalview 2.4.0 b2 (Waterhouse et al. 2009). The H. armigera CSPs were chosen for phylogenetic analysis along with CSPs from Bombyx mori, Sesamia inferens, S. exigua, Agrotis ipsilon, Spodoptera littoralis, Manduca sexta, Plutella xyllostella, and D. melanogaster, which were downloaded from NCBI. A phylogenetic tree was constructed based on amino acid sequences using the neighbor-joining algorithm as implemented in MEGA6 (Tamura et al. 2011) with default settings, and 1000 bootstrap replicates. The neighbor-joining tree was manipulated and displayed by the Interactive Tree Of Life (http://itol.embl. de/) (Letunic and Bork 2007, 2011). Quantitative Real-time PCR and Data Analysis We used qRT-PCR to study the expression levels of HarmCSPs in different tissues among moths of both sexes. The qRT-PCR was done in a Mastercycle® ep realplex (Eppendorf, Hamburg, Germany). CSP specific qRT-PCR primers were designed based on nucleotide sequences using Beacon Designer 7.7 (listed in Supplemental file S1). The H. armigera GTPbinding protein mRNA (AY957405) and glyceraldehyde-3-

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phosphate dehydrogenase mRNA (JF417983) sequences were used as reference genes (Rafaeli et al. 2007). mRNA levels were measured using a GoTaq® qPCR Master Mix (Promega) according to the manufacturer’s instructions. A blank control without template cDNA (replacing cDNA with H2O) served as the negative control. Reactions were carried out as follows: 2 min at 95 °C, followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. Reactions were followed by a melting curve analysis to detect single gene-specific peaks, and to check for the absence of primer dimers, with all primers tested. Each reaction was run in triplicate (technical replicates), with three independent biological replicates. Relative CSP quantifications were calculated using the comparative 2-△△CT method (Pfaffl 2001) for identifying relative mRNA levels. E. coli Expression and Purification of the Recombinant Protein An E. coli expression system was used to express the HarmCSPs. A pGEX-4 T-1 vector was used, and the recombinant products were N-termini tagged with glutathione S-transferase for selective purification. Signal peptides, as predicted by SignalIP 4.1 (http://www.cbs.dtu.dk/services/ SignalP/) (Emanuelsson et al. 2007), were removed to generate properly folded proteins. PCR products encoding mature proteins were amplified using gene specific primers (Supplemental file S2). The purified PCR products were ligated into the expression vector pGEX-4 T-1 using an InFusion® HD Cloning Kit (Clontech, Mountain View, CA, USA) following manufacturer’s instructions, and then the recombinant plasmids were transformed into competent E. coli BL21 cells. Positive clones were validated by PCR and sequencing. Recombinant bacterial expression and purification was performed according to Li et al. 2013a). Briefly, positive clones were incubated and induced by the addition of isopropyl-β-D1-thiogalactopyranoside to a final concentration of 1 mM, when the culture reached an OD600 =0.5–0.8. Cells were grown for an additional 6 h at 220 rpm and 37 °C, harvested by centrifugation at 8000×g for 5 min at 4 °C, and sonicated on ice. Recombinant proteins were purified from the supernatant using an affinity GSTrap FF column (GE Healthcare, Piscataway, NJ, USA). The GST tags were cleaved from the proteins using 400 μl of thrombin cleaving enzyme (1U/μl), while loaded in the GSTrap FF columns at 25 °C for 12 h. Once the cleaving reaction was complete, recombinant protein was eluted using phosphate buffered saline. Finally, the eluted proteins were desalted using a HiTrap Desalting kit (GE Healthcare), lyophilized, and stored at −80 °C until required. Competitive Fluorescence Binding Assay Emission fluorescence spectra were measured on a Hitachi F-4500 fluorescence spectrophotometer in a right-angle configuration with a 1 cm light path quartz cuvette. The protein was dissolved in

50 mM Tris–HCl buffer, pH=7.4, while all ligands used in binding experiments were added as 1 mM methanol solutions. Aliquots of 2 μM CSP solution were titrated with 1 mM 1N-phenylnaphthylamine (1-NPN) to a final concentration of 16 μM to measure CSP affinity for the fluorescent probe. The 1-NPN fluorescent probe was excited at 337 nm, with the emission spectra recorded from 360 to 500 nm in 5 nm increments. Dissociation constants for 1-NPN were calculated from Scatchard plots of the binding data. The CSP and 2 μM 1NPN solutions were titrated with each ligand to measure the affinities of the CSPs for each compound, and the corresponding fluorescence intensities were recorded. Dissociation constants (Ki) of the CSPs for each ligand were calculated from the corresponding IC50 (concentrations of ligands halving the initial fluorescence value of 1-NPN) according to the binding curves, using the following equation: Ki ¼

IC50 C1−NPN 1þ K1−NPN

In this equation, C1-NPN is the free concentration of C1NPN, and K1-NPN is the dissociation constant of the complex protein/1-NPN (Wei et al. 2008). Each reaction was repeated in triplicate. Structural Modeling ClustalX was used to create a multiple protein sequence alignment, which was drawn with ESPript (http://espript.ibcp.fr/ESPript/ESPript/) (Gouet et al. 1999). Template searches for structural modeling were obtained from the RCSB Protein Data Bank (http://www.rcsb.org/ pdb/). The crystal structure of CSPsg4 (Tomaselli et al. 2006) was used as a template to construct a 3-D structural model of HarmCSP6 using Modeller 9v7 (http://salilab.org/ modeller). Models were rendered using PyMol (http://www. pymol.org/).Statistical Analysis Data (mean ± SE) from various samples were subjected to one-way nested analysis of variance followed by a least significant difference test for mean comparisons. Two-sample analyses was performed by Student’s t-tests using SPSS Statistics 17.0 (IBM, Chicago, IL, USA). In Situ Hybridization For in situ hybridization 369 bp of digoxigenin (DIG)-labeled sense and antisense RNA probes were employed. These probes were generated using the T7/ SP6 RNA transcription system (Roche, Mannheim, Germany) following recommended protocols. To improve hybridization signals, the probes were fragmented to an average length of about 200 bp by incubation with carbonate buffer (80 mM NaHCO3, 120 mM Na2CO3, pH 10.2). Antennae were dissected from 1- to 2-d-old moths, embedded in Jung tissuefreezing medium (Leica, Nussloch, Germany) and frozen at −70 °C on the object holder. Cryosections (12 μm) of

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antennae were thaw mounted on slides coated with Vectabond (Leica) and air dried at room temperature for 20 min. After treatment with fixation solution (4 % paraformaldehyde in 0.1 M NaHCO3/Na2CO3, pH 9.5) for 30 min at 4 °C, antennae were washed for 1 min in PBS (phosphate-buffered saline: 0.85 % NaCl, 1.4 mM KH2PO4, 8 mM Na2HPO4, pH 7.1), incubated for 10 min in 0.6 % HCl, followed by PBS 2 min with 1 % Triton X100 and two 30 sec washes in PBS. Following this the slides were rinsed for 10 min in 50 % formamide/5×SSC (saline sodium citrate solution: 0.15 M NaCl, 0.015 M Na-citrate, pH 7.0). Subsequently, antennae were prehybridized with in situ hybridization solution (50 % formamide, 2×SSC, 10 % dextran sulfate, 20 mg/ml yeast tRNA, 0.2 mg/ml herring sperm DNA) containing DIGlabeled antisense RNA and incubated at 55 °C over night. Posthybridization, the slides were washed twice in 0.1×SSC for 30 min at 60 °C, rinsed shortly in TBS (tris-buffered saline: 100 mM Tris, pH 7.5, 150 mM NaCl), and incubated in 1 % blocking reagent (Roche) in TBS plus 0.03 % Triton-X100 for 30 min at room temperature, and incubated in 1 % blocking

Table 1

reagent (Roche). After three washes for 5 min in TBS plus 0.05 % Tween-20 and shortly rinsed in detection buffer (100 mM Tris, pH 9.5, 100 mM NaCl, 50 mM MgCl2), positive hybridization was visualized using nitroblue tetrazolium / 5-brom-4-chlor-3-indolyl phosphate. Finally, pictures were taken with an Olympus microscope with contrast and brightness adjusted only in Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA).

Results Identification of HamCSP Genes We identified a total of 24 putative HarmCSPs in our cDNA library using homologybased sequence analysis. We screened the 24 candidate HarmCSPs to verify their identity and to remove redundancy by sequencing the PCR products and searching each against the Nr database using BLASTX. The corresponding predicted HarmCSP proteins all belong to the OS-D superfamily with Evalues < 10−5 (Table 1). Twenty of our cDNA sequences

Blastx matches of Helicoverpa armigera Chemosensory Protein genes

Name

ORF

Acc.number

Best Blastx Match Name

Species

E-value

Identity

Acc. number

HarmCSP2 HarmCSP5 HarmCSP6 HarmCSP7 HarmCSP8* HarmCSP9* HarmCSP10*

363 384 318 336 306 387 369

AEX07265 AEB54579 AEX07267 AEX07268 AFR92092 AFR92093 AFR92094

CSP2 CSP5 CSP6 CSP7 chemosensory protein 8 chemosensory protein 9 chemosensory protein 10

Helicoverpa armigera Helicoverpa armigera Helicoverpa armigera Helicoverpa armigera Helicoverpa armigera Helicoverpa armigera Helicoverpa armigera

1.00E-82 6.0E-87 3.0E-82 6.00E-71 7.00E-67 2.00E-76 9.0E-84

100 % 100 % 100 % 97 % 100 % 100 % 100 %

AEX07265 AEB54579 AEX07267 AEX07268 AFR92092 AFR92093 AFR92094

HarmCSP11* HarmCSP12* HarmCSP13* HarmCSP14* HarmCSP15 HarmCSP16 HarmCSP17* HarmCSP18* HarmCSP19* HarmCSP20* HarmCSP21* HarmCSP22* HarmCSP23* HarmCSP24* HarmCSP25* HarmCSP26* HarmCSP27*

387 396 384 396 324 366 305 372 387 381 372 324 378 357 396 307 337

AFR92095 AFR92096 AFR92097 AFR92098 AGH20053 AGH20054 KM236059 KM236060 KM236061 KM236062 KM236063 KM236064 KM236065 KM236066 KM236067 KM236068 KM236069

chemosensory protein 11 chemosensory protein 12 chemosensory protein 13 chemosensory protein 14 chemosensory protein 15 chemosensory protein 16 chemosensory protein 11 chemosensory protein 8 chemosensory protein 11 chemosensory protein 2 chemosensory protein 2 chemosensory protein 5 chemosensory protein 11 chemosensory protein 2 chemosensory protein 9 precursor chemosensory protein 4 chemosensory protein 18, partial

Helicoverpa armigera Helicoverpa armigera Helicoverpa armigera Helicoverpa armigera Helicoverpa armigera Helicoverpa armigera Helicoverpa armigera Agrotis ipsilon Helicoverpa armigera Heliothis virescens Agrotis ipsilon Agrotis ipsilon Helicoverpa armigera Dendroctonus ponderosae Bombyx mori Aphis gossypii Helicoverpa assulta

1.0E-87 1.0E-87 3.0E-86 4.0E-88 1.0E-62 8.0E-82 2.0E-29 2.0E-66 9.0E-79 4.0E-81 6.0E-66 4.0E-64 3.0E-59 1.00E-39 1.00E-81 2.00E-55 1.00E-19

100 % 100 % 100 % 100 % 100 % 100 % 57 % 76 % 88 % 94 % 76 % 92 % 73 % 54 % 68 % 100 % 98 %

AFR92095 AFR92096 AFR92097 AFR92098 AGH20053 AGH20054 AFR92095 AGR39578 AFR92095 AAM77040 AGR39572 AGR39575 AFR92095 AGI05172 NP_001037069 AGE97643 AGH20056

HarmCSPs marked with an asterisk were identified by our laboratory

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Fig. 1 The number of Chemosensory Protein genes in different insect species, obtained from genome or transcriptome data

contained full-length open reading frames, based on sequence analysis. A comparison of the 24 sequences with all published CSPs revealed that six of the sequences, including HarmCSP2, 5, 6, 7, 15, and 16, had been previously described. The total number of putative HarmCSPs was similar to the number of CSPs identified from M. sexta (21), S. inferens (24), S. littoralis (21), B. mori (18), Chrysopa pallens (22), and Tribolium castaneum (20) (Li et al. 2013b; Zhang et al. 2013b) (Fig. 1).

Sequence Alignment and Phylogenetic Analysis All of the predicted HarmCSPs have four conserved cysteine residues except for HarmCSP27, because it was incomplete, and HarmCSP13, which contains three conserved cysteine residues (Fig. 2). The HarmCSPs display a high level of identity; pair-wise identities between the HarmCSP5, 9, 11, 12, 14, 15, 18, 19, and 23 genes were all greater than 73 %. We used 23 HarmCSPs, excluding HarmCSP27 owing to its abbreviated length, along with CSPs from other insects to construct a phylogenetic tree based on amino acid sequences. The tree reveals that all of our HarmCSPs belong to orthologous sequence groups in the other species. HarmCSP6 is tightly associated with SinfCSP19, SlitHO118383, MsexCSP11, and BmorCSP11 with 90 % bootstrap support (Fig. 3). HarmCSP Tissue Expression Profiles The abundance of HarmCSP transcripts was investigated using qRT-PCR on different cotton bollworm tissues. The HarmCSPs displayed several different expression patterns (Fig. 4), with some genes exhibiting similar expression profiles, while others showed distinct patterns. HarmCSP5 and 6 were highly expressed in antennae, with HarmCSP6 being expressed more highly in male compared with female antennae. HarmCSP18 and 23

Fig. 2 Alignment of Chemosensory Protein amino acid sequences. Predicted signal peptides are boxed, and the conserved cysteines are highlighted in blue and labeled by red pentagrams

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Fig. 3 Phylogenetic analysis of amino acid sequences of Chemosensory Proteins (CSPs) from Helicoverpa armigera and other species using the neighbor-joining method. Values at the nodes indicate bootstrap support

percentages based on 1000 replicates with branches with bootstrap values above 50 % indicated. CSPs from H. armigera are highlighted in red

were expressed predominantly in pheromone glands, with HarmCSP18 exclusively detected in this tissue. Four genes, HarmCSP9, 11, 12, and 19, were expressed in female and male proboscises at relatively high levels. The expression patterns of the other genes, HarmCSP7, 8, 13, 14, 16, and 22, were ubiquitous in most tissues at relatively high levels. Although the HarmCSP7 expression profile was ubiquitous, it was more highly expressed in

antennae than in other tissues. HarmCSP2, 10, 15, 20, 21, 24, 25, 26, and 27 were expressed highly in all other tissues, i.e., wings, legs, and abdomen. In Vitro Expression of HarmCSP Proteins We chose four HarmCSP genes (6, 9, 18, and 19) to produce in bacteria based on our qRT-PCR results, all of which were abundantly expressed in antennae, proboscises, or pheromone glands. All

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Fig. 4 Relative mRNA expression levels of the 24 HarmCSPs in different tissues of adults. The relative expression levels were normalized using GTP-binding protein and GAPDH. Error bars represent standard errors. H, head; T, thorax; Ab, abdomen; L, leg; W, wing; A, antenna; Pr, proboscis; Pg, Pheromone gland. Y axis are log

scale of relative mRNA expression level. The significant differences between different tissues of female are marked with red letters and male are marked with blue letters (P50 μM) to all of CSPs are β-caryophyllene, Farnesene, α-phellandrene, (+/−)-α-pinene, (+)-β-pinene, Ocimene, (−)Trans-caryophyllene, Ethylbenzene, Indole, Naphthalene, Cumene, Tridecane, Tetradecane, Undecane, Dodecane, Tetradecane, Octadecane, Heptadecane, Tridecane, Benzyl alcohol, 1-Hexanol, Cis-3-hexen-1-ol, Cis-2-hexen-1-ol, Geraniol, (+/−)-Linalool, Eucalyptol, α-ionol, Farnesol, 2-Heptanol, (+)-Cedrol, Linalool, Nerolidol, Trans −3-Hexen-1, Methyl anthranilate –ol, Butyl formate, Caproyl acetate, Octyl aldehyde, Cis-3-hexenyl hexanoate, Pentyl acetate, Ethyl propionate, Ethyl benzoate, Octyl acetate, Cis-3hexenyl acetate, Trans-2-Hexenyl acetate, Phenylacetaldehyde, βcyclocitra, (+)-Carvone, Damascenone, Geranylacetone, 6-Methyl-5hepten-2-one, 2-Heptanone, Decanal, (±)-Camphor, Dodecyl aldehyde, 2-Pentadecanone, Acetophenone, Hexyl butyrate, (+)-Limonene oxide, and (E3)-Hexen-1-ol, Camphene, (R)-(+)-limonene, Phenethyl alcohol, 3-Hexanol, Trans-nerolidol, Cis-3-Hexenol, Benzyl acetate, Phenethyl acetate, Geranyl acetate, Cis-3-hexenyl acetate, Ethyl butyrate, Trans-2hexenyl butyrate, Heptyl acetate, Methyl salicylate, Butyl acetate, Nonyl acetate, Isoamyl acetate, Cis-3-Hexenyl butyrate, Benzaldehyde, Hexanal, Trans-2-hexenal, Cis-3-hexenal, 3-Hexanone, 2-Hexanone, βionone

Insect CSPs belong to a divergent, multigene family. In this study, 24 HarmCSP genes were cloned and identified, including six that had been previously described. This number of putative HarmCSPs was similar to several other Lepidoptera insects, but more than those found in D. melanogaster (4), Anopheles gambiae (7) (Wanner et al. 2004), A. mellifera (6), and Acyrthosiphon pisum (13) (Zhou et al. 2010). An alignment of our HarmCSP protein sequences shows that most are between 100 to 120 amino acid residues in length, and contain four highly conserved cysteine residues, consistent with previous studies (Zhou et al. 2006). Our phylogenetic analysis shows that the HarmCSPs segregate into orthologous CSP clades with those of other Lepidoptera species, rather than into a H. armigera paralogous clade. Insect CSPs serve various functions, including chemosensation (González et al. 2009) and development (Maleszka et al. 2007), as well as other processes (Kulmuni and Havukainen 2013). Insect CSPs have been reported to bind, transport, and deliver exogenous hydrophobic chemicals to receptors through the sensillum lymph of chemosensory organs. This chemosensory function requires two components: the proteins need to act in the sensillum lymph by being expressed in chemosensory organs (Nagnan-Le Meillour et al. 2000), and the proteins need to have an internal hydrophobic binding cavity composed of α-helices that can bind the relevant chemicals (Campanacci et al. 2003; Kulmuni and Havukainen 2013; Kulmuni et al. 2013; Mosbah et al. 2003). The HarmCSPs in our study displayed various tissue expression profiles, most showing broad expression patterns, consistent with previous studies on members of this gene family in other insects (Gong et al. 2007; Gu et al. 2012; Liu et al. 2010; Qiao et al. 2013). Tissue expression patterns often can provide functional information. HarmCSP7, 8, 13, 14, 16, and 22 were ubiquitously expressed in most of tissues tested at relatively high levels. The other HarmCSPs were highly expressed in specific tissues including antennae, proboscises, pheromone glands, wings, legs, and abdomen. The broad and diverse expression patterns suggest that the CSPs play different roles during the adult stage, related to chemosensation and other processes. Those genes that are highly expressed in proboscises, HarmCSP9, 11, 12, and 19, likely play a role in gustation. Similarly, the antennae enriched genes may be involved in insect olfaction. Antennae and proboscises are the major chemosensory organs of insects, and pheromones are produced and released by specialized sex pheromone glands in H. armigera. Therefore, HarmCSP5, 6, 9, 18, and 19, which were expressed in

J Chem Ecol Fig. 8 Three-dimensional models of HarmCSP6. (a) Sequence alignment of HarmCSP6 and CSP4 from S. gregaria. Conserved residues are highlighted in white letters with a red background. Ile 73 and Trp 80 are labeled with pentagrams. The disulfide bridges are numbered 1 and 2. (b) Overall structural model of HarmCSP6

antennae, proboscises, and pheromone glands, may be involved in binding volatile compounds including sex pheromones. HarmCSP5 has been reported to play possible roles in odorant recognition, being able to strongly bind the plant volatiles 4-ehtylbenzaldehyde and 3,4-dimethlbenz aldehyde (Zhang et al. 2013a). HarmCSP6, 9, 18, and 19 were expressed using a bacterial system to perform ligand binding assays with 89 volatile compounds, including 37 from cotton (Yu et al. 2007), 10 from maize (Carroll et al. 2006; De Moraes et al. 1998; Itoh et al. 2002), 7 from tobacco (De Moraes et al. 1998; Yan et al. 2005), and 4 sex pheromone components. HarmCSP9, 18, and 19 did not show binding affinities with any of the tested compounds at a Ki>50 μM. HarmCSP6 also did not show significant binding affinity for any of the plant volatiles. However, it specifically and strongly bound the sex pheromone components (Ki5.2 μM) (Guo et al. 2012; Zhang et al. 2012). General odorant binding protein GOBP2 (Zhou et al. 2009) and CSPs (Zhang et al. 2014) have both been demonstrated to bind to sex pheromones in insects. Therefore, it is quite possible that HarmCSP6 may be involved in the recognition of female sex pheromone components. Our HarmCSP6 sequence analysis and 3-D structural modeling illustrate that I73 and W80 are homologous to I76 and W83 in CSPsg4 of S. gregaria, which are both involved in binding oleamide (Tomaselli et al. 2006). This suggests that I73 and W80 likely affect binding properties in HarmCSP6. Further studies of CSP protein/ligand complex 3-D structures are needed to show the importance of these two residues at the binding site.

J Chem Ecol Fig. 9 Topographical expression of HarmCSP6 in both female and male Helicoverpa armigera antennae. Signals were visualized using an anti-DIG antibody and color substrates. Negative control with a DIG-labeled sense probe of HarmCSP6 in female (a) and male (b) antennae. Hybridization signals in numerous cells located in or in very close proximity to long sensilla trichodea on female (c, e, g and j) and male (d, f, h and i) antennae. Scale bars represent 20 μm

The in situ hybridization experiments showed that HarmCSP6 was expressed in numerous cells located in or in very close proximity to long sensilla trichodea on the antennae of both males and females. Previous studies have demonstrated that the long sensilla trichodea are involved mainly in sex pheromone detection in lepidopteran insects (Lee and Strausfeld 1990; Liu et al. 2014). Therefore, the cellular location of HarmCSP6 is consistent with this CSP being involved in the transportation of sex pheromones. In summary, we cloned 24 CSP genes from H. armigera and showed that most HarmCSPs are ubiquitously expressed in adult tissues. CSPs serve varied functions, including chemosensation (González et al. 2009), immune response (Stathopoulos et al. 2002), moulting (Kitabayashi et al. 1998), and development (Maleszka et al. 2007), as well as

other processes (Kulmuni and Havukainen 2013). Our experiments provide evidence that HarmCSP6 may be involved in detection of the sex pheromone. Further studies are required to show fully possible roles of the CSP family in H. armigera, which may aid the design of molecules for pest management purposes.

Acknowledgment We thank Nai-Yong Liu (Nanjing Agricultural University, China) for help in 3-D structural modeling, and professer Guirong Wang and Mengbo Guo for help in the in situ hybridization experiments. This study was funded by research grants from the Ministry of Agriculture of China (2014ZX08011-002) and the National Natural Science Foundation of China (31071978). Competing Interests The authors declare no conflict of interest.

J Chem Ecol

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