Comparative Biochemistry and Physiology, Part D 13 (2015) 44–51

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Identification of candidate chemosensory genes in the antennal transcriptome of Tenebrio molitor (Coleoptera: Tenebrionidae) Su Liu 1, Xiang-Jun Rao 1, Mao-Ye Li, Ming-Feng Feng, Meng-Zhu He, Shi-Guang Li ⁎ College of Plant Protection, Anhui Agricultural University, Hefei, Anhui 230036, PR China

a r t i c l e

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Article history: Received 2 November 2014 Received in revised form 25 December 2014 Accepted 26 January 2015 Available online 2 February 2015 Keywords: Yellow mealworm beetle Antennae Transcriptome Chemosensory genes Insect olfaction

a b s t r a c t We present the first antennal transcriptome sequencing information for the yellow mealworm beetle, Tenebrio molitor (Coleoptera: Tenebrionidae). Analysis of the transcriptome dataset obtained 52,216,616 clean reads, from which 35,363 unigenes were assembled. Of these, 18,820 unigenes showed significant similarity (E-value b 10−5) to known proteins in the NCBI non-redundant protein database. Gene ontology (GO) and Cluster of Orthologous Groups (COG) analyses were used for functional classification of these unigenes. We identified 19 putative odorant-binding protein (OBP) genes, 12 chemosensory protein (CSP) genes, 20 olfactory receptor (OR) genes, 6 ionotropic receptor (IR) genes and 2 sensory neuron membrane protein (SNMP) genes. BLASTX best hit results indicated that these chemosensory genes were most identical to their respective orthologs from Tribolium castaneum. Phylogenetic analyses also revealed that the T. molitor OBPs and CSPs are closely related to those of T. castaneum. Real-time quantitative PCR assays showed that eight TmolOBP genes were antennaespecific. Of these, TmolOBP5, TmolOBP7 and TmolOBP16 were found to be predominantly expressed in male antennae, while TmolOBP17 was expressed mainly in the legs of males. Several other genes were identified that were neither tissue-specific nor sex-specific. These results establish a firm foundation for future studies of the chemosensory genes in T. molitor. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Olfaction is of vital importance for insects' survival and reproduction. Insects have an excellent sense of smell thanks to a unique olfaction system that contains a variety of chemosensory proteins that are involved in olfaction signal transduction pathways (odorant-binding proteins, OBPs; chemosensory proteins, CSPs; olfactory receptors, ORs; ionotropic receptors, IRs; sensory neuron membrane proteins, SNMPs) (Leal, 2013). OBPs and CSPs are small soluble proteins that function as shuttles to transport hydrophobic odorants through the sensillum lymph and deliver molecules that interact with receptors (Zhou, 2010). The hallmark of classic OBPs is the presence of six positionally conserved cysteine residues (Pelosi et al., 2014). These cysteines form three disulfide bridges that maintain the tertiary structure of the protein (Zhou, 2010). Knockdown of OBP genes in vivo or removal of OBPs in vitro usually leads to a decrease in the sensitivity for ORs to detect specific odorants (Xu et al., 2005; Grosse-Wilde et al., 2006; Pelletier et al., 2010). Other studies have revealed that OBPs undergo pH-independent and, more ⁎ Corresponding author at: College of Plant Protection, Anhui Agricultural University, 130 West Changjiang Road, Hefei, Anhui 230036, PR China. Tel./fax: +86 551 65786312. E-mail address: [email protected] (S.-G. Li). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.cbd.2015.01.004 1744-117X/© 2015 Elsevier Inc. All rights reserved.

intriguingly, ligand-induced conformational changes (Damberger et al., 2007; Laughlin et al., 2008; Leite et al., 2009). Therefore, the responses of receptors might be triggered by odorant molecules or by an OBP/odorant complex (Laughlin et al., 2008). CSPs, unlike OBPs, contain four positionally conserved cysteine residues that form two disulfide bridges (Pelosi et al., 2014). Some CSPs are distributed in the antennae sensillar lymph where they bind host volatiles and pheromone constituents (Dani et al., 2011; Zhang et al., 2014). Other CSPs are highly expressed in non-olfactory tissues, including pheromone glands, legs and wings, suggesting they participate in other physiological processes (Jacquin-Joly et al., 2001; Pelletier and Leal, 2011). Receptors, ORs and IRs, are key proteins located on the surface of the dendritic membrane of the olfactory sensory neurons (OSNs), which are housed in the olfactory sensilla on the antennae and maxillary palps (Touhara and Vosshall, 2009; Leal, 2013). ORs are divergent proteins with a seven-transmembrane domain but adopt a specific membrane topology with an intracellular N-terminus (Benton et al., 2006). A typical OR unit consists of one conventional OR and one highly conserved, nonconventional olfactory co-receptor (Orco) (Jones et al., 2005). Recent studies have shown that the insect OR/Orco complexes function as heteromeric ligand-gated ion channels (Sato et al., 2008). IRs are another class of odorant receptors that have a similar molecular structure to ionotropic glutamate receptors (iGluRs), but are not closely related to iGluRs according to protein sequence analysis (Benton et al., 2009). Like

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ORs, IRs are also divergent receptors. They accumulate in the sensory dendrites of the OSNs and tune to small amine-like volatile chemicals (Benton et al., 2009; Abuin et al., 2011). The SNMPs belong to the CD36 family of proteins and play a role in pheromone perception (Vogt et al., 2009). The SNMP1 subfamily is located on the membrane of pheromone-sensitive OSNs and is coexpressed with pheromone receptors, whereas the SNMP2 subfamily is expressed in the supporting cells surrounding the pheromonesensitive OSNs (Forstner et al., 2008). In the fruit fly Drosophila melanogaster and the moth Heliothis virescens, SNMP1 is essential for the detection of the pheromones 11-cis-vaccenyl acetate (cVA) and (Z)-11-hexadecenal, respectively (Benton et al., 2007; Jin et al., 2008; Pregitzer et al., 2014). However, the function of SNMP2 is still poorly understood. Knowledge of the mechanisms of insect olfaction is largely based on the identification of genes encoding chemosensory proteins in model insects (D. melanogaster, Anopheles gambiae, Bombyx mori, Tribolium castaneum, etc.) (Carey and Carlson, 2011; Hansson and Stensmyr, 2011). Chemosensory genes can be used to identify novel attractants or repellants for use in environmentally-friendly pest management (Leal et al., 2008; Carey and Carlson, 2011). The order Coleoptera contains the largest number of insect species, most of which are agricultural pests that cause great economic losses. However, apart from T. castaneum, the first coleopteran species with a sequenced genome (Tribolium Genome Sequencing Consortium, 2008), chemosensory gene families have only been identified from a limited number of nonmodel coleopteran insects (the tree killing bark beetles Ips typographus and Dendroctonus ponderosae, the emerald ash borer Agrilus planipennis, and the Japanese sawyer beetle Monochamus alternatus (Andersson et al., 2013; Mamidala et al., 2013; Wang et al., 2014). Studies examining a wider range of species will further our understanding of the chemosensory genes involved in olfactory signal pathways in this important insect guild. The yellow mealworm beetle, Tenebrio molitor (Coleoptera: Tenebrionidae), is a globally distributed pest species that lives in stored grains. Adult T. molitor males and females both produce sex pheromones that mediate behavioral responses for the opposite sex (Bryning et al., 2005). Thus, better understanding of the chemosensory gene families in T. molitor could help identify new targets for the development of novel control strategies. Previously, Graham et al. (2003) identified seven OBP isoforms in the hemolymph of T. molitor larvae and cloned nine cDNAs encoding these OBP isoforms. In this study, we performed Illumina sequencing to identify chemosensory genes in the antenna of T. molitor. We obtained 52,216,616 clean reads, assembled into 35,363 unigenes, from which we identified 19 OBPs, 12 CSPs, 20 ORs, 6 IRs and 2 SNMPs. The findings of this study provide a basis for future functional studies on these genes. 2. Materials and methods 2.1. Insects The T. molitor individuals used in the experiment were obtained from a laboratory colony maintained at the College of Plant Protection, Anhui Agricultural University, Hefei, Anhui, China. T. molitor larvae were reared on wheat bran in an insectary at 26 ± 1 °C, 60% relative humidity, and under a photoperiod of 16:8 h light:dark. Pupae were carefully collected and placed in plastic boxes covered with a layer of wheat bran. Antennae were dissected from male and female adults 1–6 days after eclosion, and stored at −80 °C until use. 2.2. RNA extraction and cDNA library construction Approximately 220 male antennae and 230 female antennae were pooled together, from which the total RNA was extracted using the SV Total RNA Isolation System (Promega, USA). The integrity of RNA

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was verified using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Following the Illumina manufacturer's instructions, the poly(A) + mRNA was purified from 20 μg total RNA using oligo(dT) magnetic beads and fragmented to short sequences. Using the NEBNext Ultra RNA Library Prep Kit (New England BioLabs, USA), first-strand cDNA was generated with a random hexamer primer, followed by second-strand cDNA synthesis. After end repairing and adaptor ligation, cDNA products were amplified by PCR and concentrated by AMPure XP beads (Beckman Coulter, USA) to create an antennae-specific cDNA library. 2.3. Sequencing, annotation and functional classification The prepared antennae cDNA library was sequenced on the Illumina HiSeq 2000 system (Illumina Inc., USA). Raw reads generated from original image data were filtered to obtain clean reads used for assembly. Transcriptome de novo assembly was performed using the CLC Genomics Workbench (version 6.0.4, CLC Bio., Aarhus, Denmark). Reads were combined to form contigs from which scaffolds were extended. Unigenes (scaffolds cannot be extended on either end) were clustered and distinct unigenes (length N350 bp) were annotated by searching against the GenBank non-redundant (nr) protein database using the BLASTX program with a cut-off E-value of 10−5. Functional classification of each unigene was performed using the automatic annotation tool Blast2GO program against the databases Gene Ontology (GO) and Cluster of Orthologous Groups (COG). 2.4. Bioinformatic analyses The signal peptides and transmembrane domains of deduced proteins were predicted using SignalP (http://www.cbs.dtu.dk/services/ SignalP/) and TMHMM (http://www.cbs.dtu.dk/services/TMHMM/), respectively. The matured T. molitor OBPs and CSPs (without signal peptide sequences) and their orthologs from other insect species were aligned using Clustal Omega (http://www.ebi.ac.uk/tools/ msa/clustalo/). A phylogenetic tree was constructed by MEGA 5.05 software using the neighbor-joining method with 1000 bootstrap replications (Tamura et al., 2011). GenBank accession numbers of genes used are listed in Table S1. 2.5. Real-time quantitative PCR Total RNA from different tissues of T. molitor adults (approximately 150 antennae from males, 140 antennae from females, 20 abdomens from males, and 100 legs from males, all obtained 1–6 days after eclosion) was isolated using the SV Total RNA Isolation System (Promega, USA). The integrity and concentration of RNA were determined using a Nanodrop 2000 spectrometer (Thermo Scientific, USA). Each RNA sample was reverse-transcribed using the PrimeScript RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Real-time quantitative PCR (qPCR) was performed on a StepOne Plus Real-time PCR System (Applied Biosystems, USA) using SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara, Dalian, China). The 20 μL reaction volume contained 10 μL SYBR Premix Ex Taq II, 0.2 μM of each primer, 10 ng cDNA template, and nuclease-free water. The thermal conditions were: one cycle of 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 20 s. A heat-dissociation protocol was added at the end of thermal cycle to confirm that only one single gene was detected by fluorescence dye, and a no-template control (NTC) was also included to detect possible contamination. Primers for qPCR are listed in Table S2. The T. molitor ribosomal protein S3 (RpS3) gene (GenBank acc. no. KJ868729) was used as a house-keeping gene. Each sample was run in triplicate from three biological replicates; relative levels of gene expression among different samples were measured by the 2−ΔΔCt method (Livak and Schmittgen, 2001).

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3.2. GO and COG classification The results of the GO analysis showed that 13,010 of the 35,363 unigenes (36.79%) corresponded to at least one GO term (Fig. 2). Among the 63 GO categories, the ‘cellular process’ (9233 unigenes, 26.11%), ‘single-organism process’ (8810 unigenes, 24.91%) and ‘metabolic process’ (8696 unigenes, 24.59%) were the three largest categories. The results of the COG analysis showed that, of the 35,363 tested T. molitor unigenes, 11,544 (32.64%) sequences had a COG classification (Fig. 3). Among the 25 COG categories, the cluster for ‘signal transduction mechanisms’ represented the largest group (1814 unigenes, 15.71%), followed by the ‘general function prediction only’ (1702, 14.74%) and ‘posttranslational modification, protein turnover, chaperones’ (1011, 8.76%).

Fig. 1. Unigene size distribution.

2.6. Data statistics 3.3. Identification of OBPs The mean values of gene expression levels were logarithmictransformed and analyzed by one-way analysis of variance (ANOVA) with the least significant difference (LSD) test, using the Data Processing System (DPS) software v9.5 (Tang and Zhang, 2013). The level of significance was set at p b 0.05.

3. Results 3.1. Unigene assembly and annotation In total, 52,216,616 clean reads were obtained from the antennal transcriptome of T. molitor. The dataset has been deposited in the NCBI Sequence Read Archive (SRA; acc. no. SRX748383). These reads were assembled into 35,363 unigenes (accumulated length of 33,020,669 bp) with an average length of 451 bp and N50 of 505 bp. The size distribution analysis indicated that the lengths of the 9318 unigenes (26.35%) were greater than 1000 bp (Fig. 1). A total of 18,820 (53.22% of all distinct sequences) unigenes resulted from the search against the NCBI nr protein database using the BLASTX algorithm (cut-off E-value of 10−5). The E-value distribution of the best match for each unigenes is shown in Fig. S1A. 58.66% of the homologous unigenes ranged between 1 × 10−5 and 1 × 10−100, whereas 41.33% had strong homology (smaller than 1 × 10−100). For species distribution, the T. molitor unigenes were best matched to those sequences from T. castaneum (90.81%; Fig. S1B). This analysis also revealed that a small amount of the T. molitor unigenes matched those from D. ponderosae, Acyrthosiphon pisum and other species.

A total of 19 putative OBP genes were identified in the T. molitor antennal transcriptome (named as TmolOBP1–TmolOBP19, Table S3). Of these, 18 sequences had full-length ORFs, whereas one sequence, TmolOBP5, had a truncation in the 5′-region. The length of the deduced TmolOBP proteins ranged from 113 to 158 amino acid residues. Signal peptide sequences were predicted in 18 TmolOBPs, but not in TmolOBP5 due to its incomplete N-terminus. Most TmolOBPs had orthologs in T. castaneum, while TmolOBP10 had a relatively low identity to an OBP from the long-horned beetle, Batocera horsfieldi (Coleoptera: Cerambycidae). TmolOBP11 and TmolOBP12 showed 89% and 100% identity to the previous characterized OBPs found in the hemolymph of T. molitor (Table S3). Sequence comparison of OBPs between the two beetle species showed that, the sequence identity percentages of four pairs of OBPs are more than 70%: TmolOBP4 and TcasOBP10 (72%), TmolOBP8 and TcasOBP17 (71%), TmolOBP9 and TcasOBP11 (83%) and TmolOBP19 and TcasOBP8 (70%). Three pairs showed identities ranging from 61 to 69%: TmolOBP1 and TcasOBP15 (61%), TmolOBP5 and TcasOBP16 (69%), and TmolOBP6 and TcasOBP16 (63%). The remaining 12 pairs showed identities lower than 59%. The insect OBPs were divided into at least five subclasses: classic (having the typical six-cysteine signature), minus-C (having lost the second and fifth cysteines), plus-C (having two additional conserved cysteines plus one proline), dimers (having two six-cysteine signatures), and atypical (having 9–10 cysteines and a long C-terminus) (Zhou, 2010). Multiple sequence alignment of the T. molitor OBPs revealed that 10 TmolOBPs (TmolOBP1–TmolOBP10) carried six

Fig. 2. Gene ontology (GO) classification of unigenes.

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Fig. 3. Clusters of Orthologous Groups (COG) classification of unigenes.

positionally-conserved cysteine residues that were divided into the classic subclass (Fig. S2A). Eight TmolOBPs (TmolOBP11–TmolOBP18) lost their second and fifth cysteine residues and were divided into the minus-C subclass. TmolOBP19 presented seven cysteine residues, six of which were conserved (Fig. S2A). The plus-C, dimers, and atypical type OBPs were not found in this study. In order to better understand the relationship of OBPs in T. molitor to those of other insect species, a neighbor-joining (NJ) tree was constructed. The NJ tree revealed a close relationship between the OBPs of T. molitor and those of T. castaneum (Fig. 4A). The 10 classic T. molitor OBPs (TmolOBP1–TmolOBP10) and TmolOBP19 were spread across several branches, while the eight minus-C type OBPs (TmolOBP11– TmolOBP18) fell into the minus-C branch (Fig. 4A). Two I. typographus OBPs (ItypOBP2 and ItypOBP10) and one D. ponderosae OBP (DponOBP2) were grouped together into the Plus-C branch (Fig. 4A). However, no T. molitor OBP fell into this branch (Fig. 4A). 3.4. Identification of CSPs In total, 12 putative CSP genes were identified (TmolCSP1– TmolCSP12, Table S3). All of these sequences had full-length ORFs and predicted signal peptides. The length of the deduced TmolCSP proteins ranged from 98 to 144 amino acid residues. The results of the BLASTX search are shown in Table S3. The deduced TmolCSPs all had high identities to known T. castaneum CSPs. Sequence comparison showed that, the percent identity of four pairs of CSPs are more than 80%: TmolCSP3 and TcasCSP8 (89%), TmolCSP4 and TcasCSP11 (80%), TmolCSP8 and TcasCSP7 (92%) and TmolCSP9 and TcasCSP10 (84%). The remaining eight pairs showed identities ranging from 69 to 75%. Multiple sequence alignment showed that all the deduced TmolCSP proteins had the characteristic hallmarks of the CSP family (i.e. four positionally-conversed cysteine residues; Fig. S2B). A phylogenetic analysis was performed to evaluate relationships among the TmolCSPs and CSPs from other beetle species. In the NJ tree it could be seen that the 12 TmolCSPs were spread across several branches and that all the TmolCSPs were clustered with at least one T. castaneum ortholog (Fig. 4B). The exceptions were TmolCSP1 and TmolCSP2, which clustered together on a single branch closely related with a branch of T. castaneum CSPs (Fig. 4B). 3.5. Identification of chemosensory receptors Bioinformatic analysis led to the identification of 20 putative ORs (Table S4). Of these, 11 sequences had full-length ORFs and encoded proteins with 3–7 transmembrane domains. Although the remaining nine sequences were incomplete cDNAs, they also encoded transmembrane proteins, indicating that these proteins were located in the membrane of the neuron cells. The co-receptor, Orco, which is highly conserved across various insect species, was also identified in the antennae of T. molitor, and shared 93% identity with the T. castaneum Orco

(Table S4). Other TmolORs showed 43–77% identities with their respective orthologs from T. castaneum (Table S4). We identified six putative IRs, including three full-length IRs and three partial IRs (Table S4). All the predicted TmolIRs had one to four transmembrane domains. The six TmolIRs showed high identities (76–91%) with their respective orthologs in T. castaneum (Table S4), but showed relatively low identities with IRs in D. melanogaster and B. mori (b 50% identities, data not shown). 3.6. Identification of SNMPs We discovered two subfamilies of SNMPs in T. molitor (TmolSNMP1 and TmolSNMP2, Table S4), as has been found in other insect species. The TmolSNMP1 and TmolSNMP2 showed 83% and 67% identity with their T. castaneum orthologs. In addition, the two TmolSNMPs both had two transmembrane domains, and both presented five positionallyconserved cysteine residues in their amino acid sequences (Fig. S3). These cysteine residues are consistent within the CD36 family members and were formed disulfide bridges in the extracellular loop (Rasmussen et al., 1998). 3.7. Tissue- and sex-specific expression of T. molitor OBPs Results of qPCR assays revealed that eight genes (TmolOBP2, 3, 5, 7, 10, 14, 15 and 16) were predominantly expressed in the antennae (Fig. 5). Of these, TmolOBP5, TmolOBP7 and TmolOBP16 were found to be specifically expressed in male antennae (Fig. 5). No gene was specifically distributed in female antennae. TmolOBP12 showed a high expression level in the abdomen, but the difference in expression levels among female antennae, abdomens and legs was not significant. TmolOBP17 was mainly expressed in leg. Other genes, such as TmolOBP1, 4, 9 and 11, were ubiquitously expressed, and were neither sex-specific nor tissue-specific (Fig. 5). 4. Discussion This study has constructed the first transcriptome dataset from the antennae of the coleopteran species, T. molitor. Prior to this study, chemosensory gene families in the order Coleoptera had been only identified from the genome of the model insect T. castaneum (Tribolium Genome Sequencing Consortium, 2008), and from the antennal transcriptome of four non-model beetle species (I. typographus, D. ponderosae, A. planipennis and M. alternatus) (Andersson et al., 2013; Mamidala et al., 2013; Wang et al., 2014). Antennal transcriptome sequencing has also been conducted for the cerambycid beetle, Megacyllene caryae, from which only ORs were identified (Mitchell et al., 2012). The three previously existing transcriptome datasets for T. molitor have been used to investigate either immune-related genetic response to pathogen challenge or the intestinal genes regulating ingestion of Bacillus thuringiensis protoxin (Oppert et al., 2012;

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Fig. 4. Phylogenetic relationships of OBPs (A) and CSPs (B) in coleopteran insect species, including Tenebrio molitor (Tmol), Dendroctonus ponderosae (Dpon), Ips typographus (Ityp) and Tribolium castaneum (Tcas). The neighbor-joining trees were constructed using MEGA software (version: 5.05, Tamura et al., 2011); bootstrap values above 50% are shown. The T. molitor OBPs (A) and CSPs (B) are highlighted in red. GenBank accession numbers of sequences used were listed in Table S1.

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Fig. 5. Expression patterns of various TmolOBP genes in different tissues, including male antennae (mAn), female antennae (fAn), male abdomens (Abd) and male legs (L). Gene expression levels in various tissues were normalized relative to that in male legs. Data are presented as the mean of three replicates (n = 3) ± SE. Different lower cases indicate significant differences (p b 0.05).

Johnston et al., 2013; Zhu et al., 2013). None have provided information on chemosensory genes. Hence, the dataset reported here substantially expands our knowledge of olfactory-related genes in coleopteran insects. We identified a suit of chemosensory genes in the T. molitor antenna: 19 OBPs, 12 CSPs, 20 ORs, 6 IRs and 2 SNMPs. The binding of odorants is the first critical step in olfactory signal transduction pathways (Leal, 2013; Pelosi et al., 2014). The binding and transportation of hydrophobic odorants are thought to be dependent mainly on OBPs (Zhou, 2010; Pelosi et al., 2014). The number of OBP genes identified for T. molitor in this study (19) was relatively low compared to other insect species whose genome or transcriptome data are available (52 OBPs in D. melanogaster, 83 in A. gambiae, 46 in B. mori, 49 in T. castaneum, and 31 in D. ponderosae) (Vieira and Rozas, 2011; Andersson et al., 2013). There are several possibilities to explain this result. First, as we only sequenced the antennal transcriptome of T. molitor adults we may have missed OBP genes expressed specifically in other olfactory or nonolfactory organs (e.g. the maxillary palp, proboscis, legs, sex pheromone gland and hemolymph) or in other developmental stages, such as the larval stage (Graham et al., 2003; Gong et al., 2014; Zhou et al., 2014). Second, we cannot rule out variability in the number of OBP genes among insect species. Finally, Illumina sequencing technology may not be powerful enough to discover all the OBP genes, especially those transcripts with extremely low abundance in the antennae. Along with OBPs, CSPs also act as carrier proteins that interact with odorants in the sensillum lymph (Pelosi et al., 2014). CSPs can bind pheromone components in Mamestra brassicae (Jacquin-Joly et al., 2001), while in Sesamia inferens CSPs bind both sex pheromones and host plant volatiles (Zhang et al., 2014). However, in the American cockroach, Periplaneta americana, CSPs are expressed in the legs and are possibly involved in the formation of the epidermis of regenerating legs (Kitabayashi et al., 1998). The numbers of CSP genes identified from insect genomes is highly variable, ranging from four in D. melanogaster to 22 in B. mori (Vieira and Rozas, 2011). We identified 12 CSPs in T. molitor

antennae, comparable to the number identified in T. castaneum (19), I. typographus (6), D. ponderosae (11) and M. alternatus (12) (Vieira and Rozas, 2011; Andersson et al., 2013; Wang et al., 2014). It is possible that some of these are involved in chemical communication; however the functional data to assess this remains to be elucidated. We identified only a small number of chemosensory receptors from the antennal transcriptome. We identified 20 ORs in T. molitor, which is far fewer than the 341 OR genes (including 79 pseudogenes) identified in the T. castaneum genome (Engsontia et al., 2008), and also fewer than identified for M. caryae (57) (Mitchell et al., 2012), I. typographus (43) and D. ponderosae (49) (Andersson et al., 2013), but more than that in A. planipennis (2) (Mamidala et al., 2013) and M. alternatus (9) (Wang et al., 2014). In insects, an OR unit consists of one conventional OR and one highly conserved, nonconventional Orco (Touhara and Vosshall, 2009). An Orco (formerly named OR83b) is an essential component indicating the location of other conventional ORs on the membrane of the OSNs, and thus, regulates insect olfaction (Jones et al., 2005). Additionally, an Orco can be used as a potential target for RNA interferencebased pest management (Zhao et al., 2011; DeGennaro et al., 2013). Orco orthologs were also identified in T. molitor. We identified six IRs in T. molitor, using D. melanogaster and T. castaneum IRs as queries to screen putative T. molitor IRs (because there are few published IR sequences for coleopteran insects). This number is far lower than that obtained for T. castaneum (23 IRs, Croset et al., 2010). It is likely that some ORs and IRs have been missed in the T. molitor transcriptome dataset. Insect SNMPs are critical for recognizing lipophilic pheromone compounds. In the moths H. virescens and Antheraea polyphemus, SNMP1 and pheromone receptor were co-expressed in the pheromoneresponsive neuron cells, while SNMP2 was expressed in the supporting cells rather than the neurons (Forstner et al., 2008). In D. melanogaster, SNMP1 play a significant role in pheromone signaling either by mediating the transfer of the pheromone cVA from the OBP to the receptor OR67d (Benton et al., 2007), or by acting as an inhibitory subunit influence on OR67d sensitivity (Jin et al., 2008). To date, SNMP

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homologs have been found in many insect species (Nichols and Vogt, 2008; Vogt et al., 2009). Here, we identified two SNMPs in T. molitor (TmolSNMP1 and TmolSNMP2), which showed high identities with SNMPs from T. castaneum, indicating a functional conservatism within these proteins. Investigation of the tissue expression patterns of chemosensory genes can help us predict their functions in T. molitor. We found that eight TmolOBPs in T. molitor were predominantly expressed in the antennae. Of these, TmolOBP5, TmolOBP7 and TmolOBP16 were specific to male antennae and may encode proteins involved in the perception of female-produced pheromones. Indeed, several insect OBPs have already been reported to bind pheromone molecules (Gong et al., 2009; Liu et al., 2010; Liu et al., 2012). Intriguingly, although adult male T. molitor also produces a sex pheromone that mediates behavioral responses for the females (Bryning et al., 2005), we did not find a female antennae-specific OBP in this study. Recognition of male pheromone in this beetle species may involve other binding proteins, such as CSPs (Zhang et al., 2014). We identified additional genes that were expressed at equivalent levels in the antennae of both sexes. These might play a role in perception of general odorants, such as grain volatiles. However, functional data are required to test this hypothesis. TmolOBP1, 4, 9, 11 and 12 were expressed both in the antennae and the abdomen, and TmolOBP17 was enriched in the legs. OBPs expressed in non-olfactory tissues have been reported in other insect species, including D. melanogaster (Galindo and Smith, 2001), Culex quinquefasciatus (Pelletier and Leal, 2011), Adelphocoris lineolatus (Gu et al., 2011) and Sogatella furcifera (He and He, 2014). OBPs distributed in the abdomen and legs are unlikely to play a direct role in olfaction but may be involved in other physiological processes. For instance, in Helicoverpa armigera, an OBP which is abundant in male seminal fluid and transferred to females is able to carry oviposition deterrents to label fertilized eggs, thus prompting the female moth away from the location where the first egg was laid (Sun et al., 2012). Similarly in D. melanogaster, two OBPs have been found exclusively on the taste bristles located on the tarsi and function in taste perception (Matsuo et al., 2007). Alternatively, Graham et al. (2003) found seven OBP isoforms in the hemolymph of T. molitor larvae, indicating these proteins may function as carriers that transfer small hydrophobic compounds through the hemolymph. Our investigation of OBP genetic profiles in T. molitor was restricted to adults and should be expanded in further studies to include larval tissues. In conclusion, we successfully constructed an antennal transcriptome dataset of T. molitor and functionally annotated a total of 18,820 unigenes. Moreover, we have identified numerous, novel chemosensory genes for this species: 19 OBPs, 12 CSPs, 20 ORs, 6 IRs and 2 SNMPs. The OBP genes we identified were differentially expressed in various tissues, indicating their distinct functions in olfactory and other physiological processes. Functional studies for these OBPs and other chemosensory genes in olfaction pathways are now needed. These studies will lead to a better understanding of the olfactory system in this beetle species. The results of this work, thus, provide important baseline information to assist in the identification of novel chemosensory genes and for gene expression profiling in coleopteran insects.

Acknowledgments This work was supported by the National Natural Science Foundation of China (31401734, 31402017) and grants from Anhui Agricultural University (2013ZR008, YJ2014-2).

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbd.2015.01.004.

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Identification of candidate chemosensory genes in the antennal transcriptome of Tenebrio molitor (Coleoptera: Tenebrionidae).

We present the first antennal transcriptome sequencing information for the yellow mealworm beetle, Tenebrio molitor (Coleoptera: Tenebrionidae). Analy...
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