Comparative Biochemistry and Physiology, Part D 16 (2015) 10–19

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Analysis of antennal transcriptome and odorant binding protein expression profiles of the recently identified parasitoid wasp, Sclerodermus sp. Chang-Xiang Zhou a, Shui-Fa Min b, Yan-LongTang c, Man-Qun Wang a,⁎ a Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China b Hubei Ecology Vocational College, Wuhan 430200, People's Republic of China c China Academy of forestry, Beijing 100091, People's Republic of China

a r t i c l e

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Article history: Received 23 March 2015 Received in revised form 28 May 2015 Accepted 24 June 2015 Available online 2 July 2015 Keywords: Antennal transcriptome Sclerodermus sp. Odorant binding protein Expression profile

a b s t r a c t We constructed an antennal transcriptome of the parasitoid wasp, Sclerodermus sp. (Hymenoptera: Bethylidae). Our analysis of the transcriptome yielded 51,830,552 clean reads. A total of 46,269 unigenes were assembled, among which 29,582 unigenes exhibited significant similarity (E-values ≤ 10−5) to sequences in the NCBI nonredundant protein database. Gene ontology (GO) and cluster of orthologous groups (COG) analyses were used for the functional classification of these unigenes. We identified ten odorant binding proteins (OBPs), ten chemosensory proteins (CSPs), eight olfactory receptors (ORs), three ionotropic receptors (IRs), six gustatory receptors (GRs), and two sensory neuron membrane proteins (SNMPs). The expression profiles of the ten OBPs were determined based on a qPCR analysis of RNA extracted from the antennae, legs, and abdomens of wingless and winged female adults and whole larvae and pupae. The highest levels of OBP5, OBP6, OBP7, and OBP9 expression were observed in the antennae of adult females. The highest levels of OBP1, OBP2, and OBP4 expression were observed in the abdomen of winged females. The highest levels of OBP3 and OBP10 expression were observed in larvae and pupae, respectively, whereas OBP8 was expressed at high levels in both larvae and pupae. Our findings establish a foundation for future studies of the molecular mechanisms of chemosensory perception in Sclerodermus sp. © 2015 Elsevier Inc. All rights reserved.

1. Introduction In insects, volatile molecules are generally detected by olfactory sensory neurons (OSNs) that are located in hair-like cuticular structures, known as sensilla, which are primarily distributed on the antennae and, to a lesser extent, on the maxillary and labial palps. The surface of sensilla have multiple pores, and the dendrites of OSNs are infiltrated by the sensillum lymph, which contains small, water soluble odorant binding proteins (OBPs) and chemosensory proteins (CSPs) (SanchezGracia et al., 2009). The cell membrane of OSNs also contains various receptor proteins that bind odor ligands (de Bruyne and Baker, 2008). Odor receptor genes expressed on insect OSNs are classified into three families (Benton et al., 2009; Touhara and Vosshall, 2009; Kaupp, 2010), which include odorant receptors (ORs), ionotropic receptors (IRs), and gustatory receptors (GRs) (Kwon et al., 2007). Sensory neuron membrane proteins (SNMPs), which are not structurally homologous with odor receptors, are also expressed in OSNs and play roles in insect olfaction. ⁎ Corresponding author at: College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China. Tel.: +86 13627126839; fax: +86 27 87280920. E-mail address: [email protected] (M.-Q. Wang).

http://dx.doi.org/10.1016/j.cbd.2015.06.003 1744-117X/© 2015 Elsevier Inc. All rights reserved.

The OBPs are believed to solubilize volatile hydrophobic molecules, enabling their delivery to odor receptors via the sensillum lymph (Sanchez-Gracia et al., 2009; Zhou, 2010; Swarup et al., 2011). The OBPs contain two or three disulfide bridges. The CSPs, which are thought to be more highly conserved than OBPs, contain two disulfide bridges only (Pelosi et al., 2006). Both OBPs and CSPs are highly abundant in the sensillum lymph (Jin et al., 2006). However, CSPs are also expressed in certain nonsensory tissues and play roles in development, molting, and leg regeneration in insects (Wanner et al., 2005). Semiochemicals are also bound by CSPs, but their roles in olfaction remain largely undetermined (Pelosi et al., 2006; R. Liu et al., 2012). Olfaction is critical for insect feeding and mating. The parasitoid wasp, Sclerodermus sp. (Hymenoptera: Bethylidae), which was recently identified in Yunnan Province in China, is an ectoparasite that preys on the larvae of the Japanese pine sawyer beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae), a major vector of the pine wood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle. Parasitic and predatory arthropods locate their herbivorous-insect prey through the simultaneous detection of a range of various herbivoreinduced plant volatiles (HIPVs) (Kessler and Baldwin, 2001; Arimura et al., 2005, 2009; Shiojiri et al., 2010). However, little is known about the sensory mechanisms involved in HIPV recognition. Female adults of Sclerodermus sp. have two wing types, including winged form and

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wingless form. More importantly, winged females are thought to disperse to long-distance hosts by flying while wingless females move mainly by walking through their complex natural environment (Zhang et al., 2012). There may be different in chemical detection between these two groups. Although the olfactory system of the parasitoids, S. pupariae (Wei et al., 2013) and S. guani (Li et al., 2015), and those of many herbivorous insects has been studied extensively (Grosse-Wilde et al., 2011; Legeai et al., 2011; Bengtsson et al., 2012; Jacquin-Joly et al., 2012; Y. Liu et al., 2012; Andersson et al., 2013; Gu et al., 2013; Zeng et al., 2013; Gong et al., 2014; Zhou et al., 2014), little is known about the olfactory mechanisms of Sclerodermus sp. In our current study, we analyzed an antennal transcriptome of Sclerodermus sp. using the Illumina HiSeq technology and de novo contig assembly to identify genes related to odor detection. We focused our bioinformatics analysis on the identification of OBP and CSP sequences, and ten OBP and ten CSP genes were identified. We also investigated the functions of these OBPs by examining their expression profiles in various tissues and developmental stages using reverse transcription and real-time polymerase chain reaction (qPCR). 2. Materials and methods 2.1. Insects Male and female adult Sclerodermus sp. were supplied by the Chinese Academy of Forestry Science and were reared at 25 °C, with 75% relative humidity and a photo period of 16:8 h (light–dark). Winged and wingless male and female adults were dissected on ice. The antennae of both males and females and the legs and abdomen of females were collected in microcentrifuge tubes. Whole larvae and pupae of both sexes were also collected. All of the specimens were stored at −80 °C. 2.2. Preparation of antennal cDNA library for transcriptome sequencing Total RNA was extracted from the antennae of 764 antennae males and females (winged form and wingless form together) and treated with DNase I. The mRNAs were isolated using magnetic beads crosslinked with Oligo (dT) polynucleotides. Illumina sequencing was performed at the Beijing Genomic Institute (Shenzhen, Guangdong, China), according to the manufacturer's instructions. The quality control steps were performed using an Agilent 2100 Bioanaylzer and an ABI Step-One-Plus Real-Time PCR System. The cDNA library was sequenced using an Illumina HiSeq 2000 system. 2.3. De novo contig assembly The raw reads were filtered to obtain high-quality de novo transcriptome sequence data. Reads with adaptor contamination were discarded. Reads with unknown nucleotides comprising N 5% of the fragment sequence were removed. Reads with a quality value ≤ 10 were discarded. The de novo transcriptome assembly was performed using the Trinity short-read assembling program (Grabherr et al., 2011). Contigs representing significant parts of individual isoforms were clustered based on gene sequence homology, and the contig clusters were assembled into unigenes. The unigene sequences were adjusted for sequence splicing, and redundant sequences were removed to obtain nonredundant unigene sequences. 2.4. Unigene annotation and classification Unigenes were aligned with known protein sequences in the NCBI Nonredundant (NCBI-NR), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Cluster of Orthologous Groups (COG) databases using BLASTX with an E-value threshold of 10−5. Each unigene

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was annotated based on the protein with which it shared the highest level of sequence similarity. The Blast2GO program (Conesa et al., 2005) was used to assign gene ontology (GO) annotations, which included molecular function, cellular component, and biological process, based on the NCBI-NR annotations. The WEGO software (Ye et al., 2006) was used to assign GO functional classifications and evaluate the distribution of gene functions at the macro level. Unigene functions were also predicted based on alignment with sequences in the COG database. The KEGG database was used to predict complex biological behaviors associated with unigene function, and pathway annotations for the unigenes were based on the KEGG annotations (Kanehisa et al., 2008). 2.5. Identification and analysis of olfactory genes The contigs of Sclerodermus sp. with homology to OBP, CSP, OR, IR, GR, and SNMP genes were verified based on additional BLAST searches of GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The SignalP, version 4.1, server (http://www.cbs.dtu.dk/services/SignalP/) was used to identify signal peptides in the candidate OBPs and CSPs (Petersen et al., 2011). TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) was used to predict transmembrane domains of candidates ORs, IRs, GRs, and SNMPs. A phylogenetic analysis of the OBPs and CSPs was performed using maximum likelihood trees constructed using MEGA5.1 with 1000 bootstrap replications (Tamura et al., 2011). First of all, we need to find the best protein models. The best model of evolution for the maximum likelihood trees was the one with the lowest BIC (Bayesian Information Criterion) score in MEGA's model test. 2.6. Quantification of OBP expression The expression patterns of ten OBPs were examined in larvae, pupae, and the antennae, legs, and abdomens of winged and wingless female adults using qPCR. Total RNA was used as template. The primers used in the qPCR analysis, which were designed using the Primer-BLAST program (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), are shown in Additional file 1. The β-actin gene was used for normalizing the level of target gene expression and correcting for sample-to-sample variation. The qPCR was performed in a reaction volume of 20 μL using the SYBR Premix Ex Taq II (Tli RNaseH Plus) master mix (Takara-Bio, Shiga, Japan) in a Bio-Rad iQ5 thermal cycler. Thermal cycling was using an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 20 s and 60 °C for 45 s. Relative quantification was performed using the comparative 2−ΔΔCT method (Livak and Schmittgen, 2001), and the data are presented as the mean ± standard error (SE) of three biological replicates performed in triplicate. Intergroup differences were evaluated using a one-way analysis of variance, and the level of statistical significance was set a p b 0.05. 3. Results and discussion 3.1. Overview of antennal transcriptome of Sclerodermus sp A total of 55,903,298 raw sequencing reads, 51,830,552 high-quality reads, and 4,664,749,680 clean nucleotides were obtained with a Q20 percentage of 98.23% (Table 1). The antenna-specific assembly resulted in 71,654 contigs and 46,269 unigenes with N50 values of 853 and 1839 bp, respectively. The mean unigene size was 1109 bp. The unigenes were grouped into 17,321 distinct clusters and 28,948 distinct singletons. For species distribution, the predicted protein sequences were compared with protein sequences derived from the draft genomes of various other species using the BLASTX algorithm (E-values ≤ 10−5). A total of 29,582 sequences were annotated based on similarity to protein sequences in other insect species. This analysis showed that 20.21%, 11.24%, and 10.73% of the protein sequences from Sclerodermus sp.

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C.-X. Zhou et al. / Comparative Biochemistry and Physiology, Part D 16 (2015) 10–19 Table 1 Summary for the antennal transcriptome of Sclerodermus sp. Total raw reads

55,903,298

Total clean reads Total clean nucleotides (nt) GC percentage Q20 percentage Total number of contigs Mean length of contigs N50 of contigs Total number of unigenes Mean length of unigenes N50 of unigenes Dinstinct clusters Distinct singletons

51,830,552 4,664,749,680 41.03% 98.23% 71,654 417 853 46,269 1109 1839 17,321 28,948

and Nasonia vitripennis. Similar trends in protein similarity were reported for the transcriptome analyses of the parasitoid wasps, Cotesia vestalis and Spalangia endius (Nishimura et al., 2012; Zhang et al., 2014). A total of 14,170 unigenes were assigned to at least one of the GO categories. As shown in Fig. 2, the distribution of unigenes annotated as molecular function showed that the genes expressed in the antennae were primarily related to binding activity (6545 unigenes, 46.19%), whereas 1236 (8.72%) of the unigenes were involved in transporter activity. Similar trends in GO annotation were also observed in the transcriptomic analyses of C. vestalis and N. vitripennis (Nishimura et al., 2012; Pannebakker et al., 2013). The COG database considers the phylogenetic relationships of protein sequences from 66 genomes of bacteria, plant, and animal species. As shown in Fig. 3, a total of 11,603 of the unigenes of Sclerodermus sp. were grouped into 25 functional COG clusters (E-value ≤ 10− 5). The largest category was general function prediction only (5232; 45.09%), which was followed by translation, ribosomal structure, and biogenesis (2087; 17.99%); replication, recombination, and repair (1916, 16.51%); and transcription (1831; 15.78%).

3.2. Identification of putative chemosensory genes

Fig. 1. Proportional homology distribution among other species based on the best BLAST hits against the NR database of Sclerodermus sp. antennal transcriptome.

were orthologs of proteins in the leaf-cutter bee, Megachile rotundata; the ant, Harpegnathos saltator; and the bumble bee, Bombus impatiens, respectively, all of which are hymenopterans (Fig. 1). The protein sequences from Sclerodermus sp. also shared similarity with proteins from other hymenopterans, including Camponotus floridana, Apis florea, Bombus terrestri, Acromyrmex echinatior, Apis mellifera, Solenopsis invicta,

We tentatively identified ten OBPs and ten CSPs in Sclerodermus sp. (Table 2). No OBP or CSP sequence from Sclerodermus sp. has been reported in the NCBI databases. Twenty-nine OBPs and 12 CSPs were identified in a study of the antennal transcriptome of the host species, M. alternatus, and 23 OBPs and 7 CSPs were identified in a transcriptomic analysis of the parasitoid, D. helophoroides (Wang et al., 2014). Of the 10 OBPs and 10 CSPs identified, SspOBP10 and SspCSP10 consisted of incomplete coding sequences. We also identified eight ORs, three IRs, and six GRs in Sclerodermus sp. (Table 3), among which SspOR1, SspIR1, and SspGR1 likely represented full-length mRNA sequences. Two transcripts encoding putative SNMPs were also identified, among which only SspSNMP1 likely represented a full-length mRNA sequence (Table 3). First, we compared the numbers of OBPs and CSPs identified in Sclerodermus sp. with those of M. alternatus and D. helophoroides which were reported from the transcriptome analysis and the numbers of the former were clearly lower than the latter (Wang et al., 2014). Because Sclerodermus sp. and D. helophoroides are the key enemies of M. alternatus in China and comparative genomics might be useful to

Fig. 2. Gene ontology (GO) classification of Sclerodermus sp. antennal transcriptome. Unigenes were classified into three main categories: biological process, cellular component, and molecular function. The left y-axis indicates the percentage of a specific category of genes in each main category. The right y-axis indicates the number of genes in the same category.

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Fig. 3. COG functional classification of Sclerodermus sp. antennal transcriptome.

learn the role of OBPs in molecular adaptations of the M. alternatus and their natural enemies' olfactory systems, then we compared the numbers of OBPs and CSPs with those reported from the genomic analysis of the model beetle Tribolium castaneum which is a powerful model organism for the study of generalized insect development, and comparison revealed that the numbers of the former were substantially lower than the latter (49 OBPs and 20 CSPs) (Richards et al., 2008). It may therefore be misleading to compare the number of insect genes identified in a genomic analysis with the number of different transcripts identified in a specific tissue at a specific life stage. A total of 90 OBP genes, 225 intact OR genes, and 47 intact GR genes have been reported from

the genomic data of parasitoid wasp, N. vitripennis, which is genetically similar to Sclerodermus sp. (Robertson et al., 2010; Vieira et al., 2012), comprising the largest collection of OBPs indentified in an insect thus far. We also made comparisons with two social insects because they all have more complex social behaviors than parasitoid wasps. By contrast, 21 OBP and six CSP genes have been reported from the genome of the honeybee, A. mellifera (Foret and Maleszka, 2006; Foret et al., 2007), and only 12 OBP genes from the genome of the fire ant, S. invicta (Wurm et al., 2011). Alignment of the amino acid sequences of the ten CSPs revealed four conserved cysteine residues (Fig. 4), which is consistent with CSPs

Table 2 Sequences information of OBPs and CSPs in Sclerodermus sp. Gene name

Accession number

ORF(aa)

SP(aa)

SspOBP1 SspOBP2 SspOBP3 SspOBP4 SspOBP5 SspOBP6 SspOBP7 SspOBP8 SspOBP9 SspOBP10 SspCSP1 SspCSP2 SspCSP3 SspCSP4 SspCSP5 SspCSP6 SspCSP7 SspCSP8 SspCSP9 SspCSP10

KP963686 KP963687 KP963688 KP963689 KP963690 KP963691 KP963692 KP963693 KP963694 KP963695 KP963706 KP963707 KP963708 KP963709 KP963710 KP963711 KP963712 KP963713 KP963714 KP963715

138 142 145 136 133 139 143 150 143 70 119 120 122 125 115 124 129 130 130 98

17 20 24 18 18 21 22 23 23 20 20 16 21 20 16 16 19 21 18

Note: ORF, open reading frame; SP, signal peptides; aa, amino acid.

Homology search with known proteins Identity

Species

E-value

Protein ID

40% 98% 38% 50% 98% 43% 43% 94% 49% 56% 46% 60% 68% 64% 77% 59% 98% 52% 54% 58%

Argyresthia conjugella Sclerodermus guani Apis mellifera Sclerodermus guani Sclerodermus guani Megachile rotundata Harpegnathos saltator Apis mellifera Camponotus floridanus Acromyrmex echinatior Polistes dominula Polistes dominula Apis cerana cerana Camponotus japonicus Apis cerana cerana Dendroctonus ponderosae Sclerodermus guani Batocera horsfieldi Apis cerana cerana Schistocerca gregaria

1e-26 1e-97 6e-19 3e-34 1e-88 6e-29 1e-33 3e-77 1e-32 9e-20 3e-27 1e-34 2e-54 1e-42 7e-59 2e-39 2e-88 7e-38 2e-40 1e-24

AFD34173.1 ABE68831.1 NP_001164515.1 ABE68830.1 ABE68830.1 XP_003708550.1 EFN77138.1 XP_006566011.1 EFN71465.1 EGI63313.1 AAP55719.1 AAP55719.1 AFQ07771.1 BAD83397.1 ACI03403.1 AFI45003.1 ABE68832.1 AEC04844.1 AFQ07769.1 AAC25403.1

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Table 3 Sequences information of ORs, IRs, GRs, and SNMPs in Sclerodermus sp. Gene name

Accession number

ORF(aa)

TMD (No.)

SspOR1 SspOR2 SspOR3 SspOR4 SspOR5 SspOR6 SspOR7 SspOR8 SspIR1 SspIR2 SspIR3 SspGR1 SspGR2 SspGR3 SspGR4 SspGR5 SspGR6 SspSNMP1 SspSNMP2

KP963696 KP963697 KP963698 KP963699 KP963700 KP963701 KP963702 KP963703 KP963683 KP963684 KP963685 KP963677 KP963678 KP963679 KP963680 KP963681 KP963682 KP963704 KP963705

480 348 121 120 279 152 115 180 825 150 360 371 221 208 149 194 121 531 91

7 6 2 2 3 2 0 2 3 1 3 7 2 4 2 4 1 2 0

Homology search with known proteins Identity

Species

E-value

Protein ID

84% 37% 40% 36% 26% 33% 38% 46% 69% 64% 60% 85% 61% 56% 63% 80% 40% 59% 53%

Cephus cinctus Nasonia vitripennis Harpegnathos saltator Acromyrmex echinatior Nasonia vitripennis Acromyrmex echinatior Microplitis demolitor Harpegnathos saltator Apis dorsata Acromyrmex echinatior Acromyrmex echinatior Harpegnathos saltator Harpegnathos saltator Megachile rotundata Apis florea Camponotus floridanus Apis dorsata Apis cerana cerana Megachile rotundata

0 3e-69 2e-22 1e-16 1e-22 3e-11 1e-14 8e-34 0 5e-57 4e-144 0 9e-85 8e-61 1e-56 4e-110 1e-16 0 5e-25

AGS43074.1 NP_001164395.1 EFN79925.1 EGI69848.1 NP_001177702.1 EGI58733.1 XP_008548430.1 EFN78910.1 XP_006621137.1 EGI70317.1 EGI70677.1 EFN86817.1 EFN83533.1 XP_003701221.1 XP_003692595.1 EFN68708.1 XP_006624554.1 AGC91908.1 XP_003703897.1

Note: ORF, open reading frame; aa, amino acids; TMD: transmembrane domain.

in other insects. The OBP family is divided into five major subclasses, which include classic (6 cysteines), minus-C, plus-C, dimers, and atypical OBPs (Gong et al., 2009; Kirkness et al., 2010; Vieira and Rozas, 2011). Alignment of the amino acid sequences of the ten OBPs from Sclerodermus sp. indicated that all nine of the full-length transcripts (SspOBP1–9) were classic OBPs (Fig. 5). With the exception of

SspOBP10, all of the OBPs and CSPs from Sclerodermus sp. contained a signal peptide (Table 2). According to the BIC score, the WAG model was used for the phylogenetic analysis of the OBPs and CSPs of Sclerodermus sp. The rate among sizes for the OBPs and CSPs were gamma distributed with invariant sites and gamma distributed, respectively. As shown in Fig. 6, a

Fig. 4. Alignment of amino acid sequences of CSPs in Sclerodermus sp.

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Fig. 5. Alignment of amino acid sequences of OBPs in Sclerodermus sp.

Fig. 6. Maximum-likelihood dendrogram based on protein sequences of candidate OBPs. Included are OBPs from Sclerodermus sp. (Ssp), Monochamus alternatus (Malt), Dastarcus helophoroides (Dhel), Acromyrmex echinatior (Aech), Apis cerana (Acer), Apis mellifera (Amel), Sclerodermus guani (Sgua), Solenopsis invicta (Sinv), and Tribolium castaneum (Tcas).

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phylogenetic tree of the OBPs was constructed based on orthologs from M. alternatus (the host), Dastarcus helophoroides (another parasitoid of M. alternatus), and certain other species in consequence of their high protein sequence similarity. The clustering of the OBPs was based on significant boot-strap values (50% cutoff) and 1000 bootstrap replicates. As shown in the dendrogram, almost all of the OBPs of Sclerodermus sp. clustered with one orthologous protein from a hymenopteran. Some of the OBPs and CSPs of Sclerodermus sp. were orthologous with proteins from S. guani, which might be expected because they belong to the same genus. The SspOBP2 protein clustered with SguaOBP2, and SguaOBP1 clustered with SspOBP4, SspOBP5, and SspOBP6. However, the OBPs of Sclerodermus sp. exhibited low levels of homology to OBPs from M. alternatus and D. helophoroides. The phylogenetic tree of the CSPs is shown in Fig. 7. The SspCSP7 protein was orthologous to SguaCSP1. The CSPs of Sclerodermus sp. exhibited low levels of homology to CSPs of M. alternatus and D. helophoroides, with the exception of SspCSP5, which was orthologous to MaltCSP1 and MaltCSP4, which indicate that these proteins might share similar structures and functions (Fitch, 1970). The function of these CSPs and the phylogenetic relationship between M. alternatus and Sclerodermus sp. require further study. The accession numbers for the amino acid sequences of the OBPs and CSPs included in the phylogenetic analyses are shown in Additional file 2. 3.3. Expression profiles of OBPs We conducted a qPCR analysis of the expression of the OBPs of Sclerodermus sp. in different tissues and development stages in an

attempt to characterize the functions of these proteins. The OBP5, OBP6, OBP7, and OBP9 transcripts were expressed at high levels in the antennae (Fig. 8). Of these, OBP9 was highly expressed in the antennae of wingless females in comparison to winged female antennae. The expression pattern of OBP5 was similar to that reported for its ortholog, SguaOBP1, which exhibits antenna-specific expression in female S. guani (Li et al., 2011). The OBP5, OBP6, OBP7, and OBP9 proteins might play roles in the perception of certain HIPVs for locating hostinsect species (Nishimura et al., 2012). The OBP9 might have a main function in host-searching behavior of Sclerodermus sp. wingless females. However, functional data are required to demonstrate this relationship. High levels of OBP1, OBP2, and OBP4 expression were observed in the abdomens of winged females only. The difference between the levels of OBP1 expression in the abdomen of winged females was not significantly different than that in the antennae of wingless females. The difference between the level of OBP2 expression in the abdomen and antennae of winged female and those of wingless females was not significant. The difference between the levels of OBP4 expression in the abdomen of winged females was not significantly different than that in larvae. The expression of OBPs in nonolfactory tissues has been reported in other insect species, including N. lugens (Zhou et al., 2014), Sogatella furcifera (He and He, 2014), and Tenebrio molitor (Liu et al., 2015). Although abdominal OBPs are unlikely to play a direct role in olfaction, they may be involved in other physiological functions related to reproduction. Studies showed that female wasps of sibling species of Sclerodermus paralyze the larvae or pupae by injecting venom and then lay eggs on the host, once they detected the hosts (Li et al., 2015). So we infer that OBP1, OBP2, and OBP4 play a role in the oviposition behavior of Sclerodermus sp. winged females.

Fig. 7. Maximum-likelihood dendrogram based on protein sequences of candidate CSPs. Included are CSPs from Sclerodermus sp. (Ssp), Monochamus alternatus (Malt), Dastarcus helophoroides (Dhel), Apis cerana (Acer), Adelphocoris lineolatus (Alin), Apolygus lucorum (Aluc), Apis mellifera (Amel), Antheraea yamamai (Ayam), Batocera horsfieldi (Bhor), Nilaparvata lugens (Nlug), Nylanderia pubens (Npub), Sclerodermus guani (Sgua), and Tribolium castaneum (Tcas).

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Fig. 8. Expression profiles of Sclerodermus sp. OBPs in different development stages and in different tissues of female adults, including larvae (LV), pupae (PU), wingless female antennae (LA), legs (LL), and abdomens (LB), winged female antennae (DA), legs (DL), and abdomens (DB). Gene expression levels were normalized relative to that in the larvae. Data are presented as the mean ± SE (n = 3). Different lower cases indicate significant differences (p b 0.05).

Although OBP3, OBP8, and OBP10 displayed no tissue-specific expression in adult female Sclerodermus sp, the expression of these transcripts varied between the different developmental stages. A high level of OBP3 expression was observed in larvae only. The expression

pattern of OBP3 was similar to that reported for OBP1 and OBP10 in Sitodiplosis mosellana (Gong et al., 2014). High level OBP10 expression in Sclerodermus sp. was observed in pupae only. High level expression of OBP8 in Sclerodermus sp. was observed in both in larvae and pupae,

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but the level of expression in pupae was significantly higher than that in larvae. Similar larva- and pupa-specific patterns of expression have been reported in other insects. In A. mellifera, OBP13 is expressed at high levels in late-instar larvae and pupae (Foret and Maleszka, 2006). In Bombyx mori, the level of OBP31 expression rises gradually, reaching its highest level in late-stage pupae (Gong et al., 2009). Larvae of the sibling species S. guani are encased in the host and complete their development until adult emergence (Li et al., 2015). Larvae and pupae of the new species Sclerodermus sp. have the same biological behaviors through observation (Zhang et al., 2012). Previous studies have suggested that OBPs allow insects to discriminate between odorants through the recognition of groups ligands with similar molecular structures (Rützler and Zwiebel, 2005). Therefore, OBPs exhibiting larva-specific expression might reflect differences in the olfactory requirements of larvae and adults. Although pupae are dormant, pupa-specific OBP expression in Sclerodermus sp. may mediate responses to ligands that are important for pupae's metamorphosis and development. 4. Conclusions Our analysis of an antennal transcriptome of Sclerodermus sp. identified ten OBPs, ten CSPs, eight ORs, three IRs, six GRs, and two SNMPs. The expression profiles of the various OBPs and CSPs revealed differences in both tissue- and development stage-specific expression, which suggested distinct functions in olfaction and other physiological processes. Future studies of the binding characteristics of these OBPs are warranted to determine their roles in the detection of HIPVs released from pine trees infested with M. alternatus. Our findings represent an important contribution to genetic resources for future studies of Sclerodermus sp. and further our understanding of odorant reception in parasitoid wasps. Acknowledgments This study was supported and funded by the National Natural Science Foundation of China (31230015), Program for New Century Excellent Talents in University (NCET-11-0649) and Excellent Youth Foundation of Hubei Scientific Committee (2011CDA088). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbd.2015.06.003. References Andersson, M.N., Grosse-Wilde, E., Keeling, C.I., Bengtsson, J.M., Yuen, M.M.S., Li, M., Hillbur, Y., Bohlmann, J., Hansson, B.S., Schlyter, F., 2013. Antennal transcriptome analysis of the chemosensory gene families in the tree killing bark beetles, Ips typographus and Dendroctonus ponderosae (Coleoptera: Curculionidae: Scolytinae). BMC Genomics 14, 198. Arimura, G.-i., Kost, C., Boland, W., 2005. Herbivore-induced, indirect plant defences. Biochim. Biophys. Acta 1734, 91–111. Arimura, G.-i., Matsui, K., Takabayashi, J., 2009. Chemical and molecular ecology of herbivoreinduced plant volatiles: proximate factors and their ultimate functions. Plant Cell Physiol. 50, 911–923. Bengtsson, J.M., Trona, F., Montagne, N., Anfora, G., Ignell, R., Witzgall, P., Jacquin-Joly, E., 2012. Putative chemosensory receptors of the codling moth, Cydia pomonella, identified by antennal transcriptome analysis. PLoS One 7, e31620. Benton, R., Vannice, K.S., Gomez-Diaz, C., Vosshall, L.B., 2009. Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell 136, 149–162. Conesa, A., Gotz, S., Garcia-Gomez, J.M., Terol, J., Talon, M., Robles, M., 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics (Oxford) 21, 3674–3676. de Bruyne, M., Baker, T.C., 2008. Odor detection in insects: volatile codes. J. Chem. Ecol. 34, 882–897. Fitch, W.M., 1970. Distinguishing homologous from analogous proteins. Syst. Zool. 19, 99–113. Foret, S., Maleszka, R., 2006. Function and evolution of a gene family encoding odorant bindinglike proteins in a social insect, the honey bee (Apis mellifera). Genome Res. 16, 1404–1413. Foret, S., Wanner, K.W., Maleszka, R., 2007. Chemosensory proteins in the honey bee: insights from the annotated genome, comparative analyses and expressional profiling. Insect Biochem. Mol. Biol. 37, 19–28.

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