Antennal transcriptome analysis and expression profiles of odorant binding proteins in two woodwasps, Sirex noctilio and S. nitobei (Hymenoptera: Siricidae)

The olfactory system is the foundation of insect behavior. Odorant binding proteins (OBPs) are key components of the insect olfactory system. The woodwasp Sirex noctilio Fabricius is a major quarantine pest worldwide that was first discovered in China in 2013 and mainly harms members of the Pinus genus. S. nitobei Matsumura is a native species in China and is closely related to S. noctilio. To gain insights into the olfactory mechanisms of these two woodwasp species, olfactory genes were identified using antennal transcriptome analysis. We also analyzed the expression profiles of OBPs with RT-qPCR. From our transcriptome analysis, 16 OBPs, 7 chemosensory proteins (CSPs), 41 odorant receptors (ORs), 8 gustatory receptors (GRs), 13 ionotropic receptors (IRs), and one sensory neuron membrane protein (SNMP) were identified in S. noctilio, while 15 OBPs, 6 CSPs, 43 ORs, 10 GRs, 16 IRs, and 1 SNMP were identified in S. nitobei. Most of the olfactory genes identified in two species were homologous. However, some species-specific olfactory genes were identified in the antennal transcriptomes, including SnocOBP13, SnocCSP6, SnocOR26, SnitGR9, and SnitIR17. In total, 14 OBPs (7 in S. noctilio and 7 in S. nitobei) were expressed primarily in the antennae of the two woodwasps. SnocOBP11 and SnitOBP11 were highly expressed in antennae and were also clearly expressed in the external genitalia. SnocOBP3 is highly expressed in the genitalia of females, and SnocOBP7 and SnitOBP7 are highly expressed in the genitalia of males. Meanwhile, SnocOBP10 was specifically expressed in male heads. ORFs, signal peptides, and a cysteine sequence pattern of C1-X5-8-C2-X18-C3-X2-C4 (Additional file 4). The expression values (FPKM) of 4 SnocCSP s and 5 SnitCSP s were greater than 1, while 1 SnocCSP and 3 SnitCSP s displayed expression values greater than 100, indicating that these genes are highly expressed in woodwasp antennae. ORFs, Open frame; FPKM, fragments per kilobase per million reads; RT-qPCR, PCR;

3 which are involved in odor recognition. A few OBPs were mainly expressed in the external genitals or heads and exhibited an obvious sex bias, which may indicate that the external genitals and heads are able to recognize sex pheromones or plant volatile compounds as a part of normal behaviors such as feeding, mating, or spawning. Our study provides key insights regarding the mechanism of interactions between the insect olfactory system and specific odor molecules.

Background
The woodwasp Sirex noctilio Fabricius (Hymenoptera: Siricidae) is native to Europe, Asia, and North Africa, and is attracted to dead or dying pines [1][2][3]. Due to increased human movement and trade, woodwasps have spread to Oceania, Africa, North America, and South America and have become a globally invasive insect species [4]. Because of the lack of competing species and natural predators, S. noctilio has had a major economic impact on various pines in invaded areas [4]. In August 2013, S. noctilio was first found in Heilongjiang and then in Liaoning, Jilin, and Inner Mongolia, China [5]. In contrast, S.
nitobei Matsumura, a species closely related to S. noctilio, is native to China, Japan, and North Korea. It has posed a hazard on ancient and debilitated pines such as the Pinus tabuliformis in Xiangshan Park, Beijing, China [6] and is a significant threat to other pine species such as P. armandi, P. tabuliformis, and Larix spp. [7]. In China, both species also harm P. sylvestris var. mongolica Litv. [8].
As wood-boring insects, woodwasps feed on wood during the larval period of their development. Adult woodwasps do not feed and only live for approximately a week [9][10].
Female adults tend to attack stressed or weakened pines, where they lay eggs and inject toxic mucus and symbiotic fungus into the host [2,11]. The affected pine trees fall into decline and display symptoms such as resinosis, interior blue staining, premature senescence, reduced growth rates, and death [12]. 4 In order to reduce the spread of and damage inflicted by woodwasps, it is important to develop effective detection tools to monitor their populations. Trap trees treated with herbicide or girdling have been used to monitor and survey S. noctilio populations [3,13].
Weak P. tabuliformis trees have been harvested for use as trap trees for attracting S.
nitobei [6]. The trap-tree method has been found to be effective but is expensive and difficult to implement. Kairomone (plant volatiles) lure traps are the most effective in areas where S. noctilio populations are large, but the traps are known to be ineffective in areas with small populations, such as newly infested areas in China [14]. Pheromones are also commonly used to develop attractants, and several pheromone compounds for S.
Insects use their olfactory systems to sense odors and changes in the environment and thus, to adjust behaviors such as locating hosts for food, mating, and spawning [18]. In this study, we explore the woodwasp olfaction system, particularly, olfactory proteins and their expression profiles in antennae. The antenna is the most important organ for olfactory recognition and sensing of pheromones or plant volatiles. There are multiple olfactory sensilla distributed on insect antennae, which house olfactory sensory neurons (OSNs). Odor molecules pass through pores on the sensilla and enter the sensillum lymph [19][20]. It has been thought that odorant binding proteins (OBPs) and chemosensory proteins (CSPs) in the lymph can recognize, bind, and transport odor molecules. The OBP/CSP-odor molecule complexes then interact with chemosensory receptors, which are located in the dendritic membrane of OSNs [21][22]. These receptors convert the chemical signals into electrophysiological signals and transmit these signals to the central nervous system of insects through axons [23][24]. These signals are integrated in the insect brain to produce behavioral instructions for insects to respond accordingly [25]. At the same time, odor molecules are degraded by odorant-degrading enzymes (ODEs) [26][27].
OBPs are soluble proteins with low molecular weights and are mainly expressed in the lymph of antenna sensilla [28][29]. Some OBPs exhibit specific binding with hydrophobic odorant molecules and can deliver the molecules to membrane-binding receptors [30][31][32][33].
Within the classic OBP subtype, there are 2 special OBP members, the pheromone binding proteins (PBPs) and the general odorant binding proteins (GOBPs). PBPs have been found to be specific affinity proteins and are involved in sex pheromone recognition [28,38]. GOBPs achieve their function by binding with general odors such as plant volatiles [39].
CSPs have 4 conserved cysteine residues, fewer than are found in OBPs, and they bind far more odors than OBPs [29,40]. In most insects, CSPs are widely expressed in both olfactory-related organs and non-olfactory organs, which may indicate that the function of CSPs is not limited to transport of odor molecules in olfaction but also includes growth and development [41,42].
Chemosensory receptors are transmembrane proteins and include odorant receptors (ORs), ionotropic receptors (IRs), gustatory receptors (GRs), and sensory neuron membrane proteins (SNMPs). ORs are seven-transmembrane-domain proteins that sense plant volatile odorants [43] as well as pheromones (as pheromone receptors [PRs]) [44][45]. A highly conserved OR, ORco [46], is involved in the localization of ORs to ORN (olfactory receptor neurons) dendrites, enhances odorant responsiveness without altering ligand specificity, and forms complexes with other ORs [23,47]. GRs are also seven-6 transmembrane-domain proteins and transport gustatory signals. Multiple GRs have been implicated in the detection of sweet tastes, bitter tastes, and CO2 [48][49]. IRs evolved from ionotropic glutamate receptors (iGluRs) and are conserved in insects [50]. In Drosophila, the majority of IRs have been identified as receptors for amines and acids, whereas ORs are receptors for esters and alcohols [51]. Two slowly evolving receptors, IR8a and IR25a, have been identified as co-receptors that can form complexes with other, more divergent IRs and direct their function as ligand-gated cation channels [52]. SNMPs are membrane proteins of insect olfactory neurons that are homologous with the vertebrate CD36 family and are thought to function as receptors of odorant binding proteins and mediate ligand delivery to chemosensory receptors [53][54][55].
In this study, we performed a preliminary exploration of the olfactory systems of two species of woodwasps, S. noctilio and S. nitobei. Ours is the first description of differential expression profiles of OBPs between different tissues and between sexes. We identify genes encoding olfactory proteins via analysis of the antennal transcriptomes of S. noctilio and S. nitobei and measure the transcript expression of important OBP genes in different tissues of both male and female adults of the two woodwasps using a quantitative real-time PCR method. We found that the olfactory genes of the two woodwasp species are extremely similar, and many are homologous. However, some species-specific olfactory genes were identified in both S. noctilio and S. nitobei. In total, 14 OBPs (7 in S. noctilio and 7 in S. nitobei) were primarily expressed in the antennae of two woodwasps. Among them, SnocOBP11 and SnitOBP11 not only have high expression in antennae, but also display notable expression in the external genitalia. We also observed several OBPs that are specifically expressed in the external genitals ( SnocOBP3 is expressed mainly in the female genitalia, while SnocOBP7 and SnitOBP7 are expressed mainly in male genitalia) or the head (SnocOBP10 is expressed mainly in the male head). Taken together, our findings reveal 7 molecular mechanisms driving the function of the woodwasps olfactory system.

Results
Transcriptome sequencing and sequence assembly Using transcriptome sequencing, a total of 174,174,820 and 168,012,792 raw reads were obtained from male and female antennae, respectively, of S. noctilio, and a total of 165,394,906 and 164,334,008 raw reads were obtained from male and female antennae, respectively, of S. nitobei (Table 1). By removing low-quality and trimmed reads less than 20 nt in length, 168,575,526 and 164,447,898 clean reads were obtained for male and female S. noctilio, respectively, and 161,515,996 and 160,823,260 clean reads were obtained for male and female S. nitobei, respectively, to be used for de novo assembly (

Chemosensory proteins
We identified 7 SnocCSPs and 6 SnitCSPs in the antennal transcriptomes of the two woodwasp species (Additional file 1, Table S2) displayed expression values greater than 100, indicating that these genes are highly expressed in woodwasp antennae.

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As with OBPs, a phylogenetic tree was used to show the evolutionary relationships between insect CSPs (Fig. 4). In the phylogenetic tree, most SnocCSPs and SnitCSPs clustered with other Hymenopteran CSPs.

Odorant receptors
We identified 41 and 43 ORs in the S. noctilio and S. nitobei antennal transcriptomes, respectively (Additional file 1, Table S3). Two woodwasp OR transcripts were identified as odorant co-receptors, and designated as SnocORco and SnocORco, respectively.
In total, 28 SnocORs and 30 SnitORs were comprised of greater than 350 amino acids and contained complete ORFs, indicating that they are nearly full-length. Using the TMHMM Server, we predicted the presence and location of transmembrane helices in the protein sequences. We found that 4 full-length SnocORs (SnocORco, SnocOR5, SnocOR8, and SnocOR30) and 3 full-length SnitORs (SnitORco, SnitOR8, and SnitOR30) possess 7 transmembrane helices. No transmembrane helices were predicted in SnocOR32, SnocOR33, SnitOR31, and SnitOR32, which is likely due to short fragments and incomplete reading frames. Four ORs (SnocOR18, SnocOR30, SnitOR18 and SnitOR30) displayed a >10fold difference in expression between males and females. The difference in the expression levels between the sexes of these 4 genes suggests that they play a role in identifying gender-related odors.
A phylogenetic tree was used to analyze the evolutionary relationships between insect ORs.
Most SnocORs and SnitORs clustered together (Fig. 5), and 2 species-specific lineages were identifed in the tree. The ORco lineage contains SnocORco and SnitORco (1.00 bootstrap support value), which further confirms that these two OBPs are ORcos.

Sensory neuron membrane proteins
We identified one SNMP in S. noctilio (SnocSNMP1) and one in S. nitobei (SnitSNMP1) (Additional file 1, Table S4). SnocSNMP1 and SnitSNMP1 are predicted to possess 2 transmembrane regions, which may indicate that SnocSNMP1 and SnitSNMP1 are full-length genes. The expression values (FPKM) for SnocSNMP1 and SnitSNMP1 were both found to be greater than 100, indicating that Sirex SNMPs are highly expressed in antennae.
SNMPs are considered to be highly conserved in holometabolous insects, but SNMP1 and SNMP2 which are members of different subfamilies, clustered separately in disparate lineages. In our phylogenetic tree, SnocSNMP1 and SnitSNMP1 clustered in the SNMP1 lineage with a bootstrap support value of 1.00 (Fig.6).

Gustatory receptors
We identified 8 and 10 GRs in the S. noctilio and S. nitobei antennal transcriptomes, respectively (Additional file 1, Table S5). Using BLASTX sequence alignment, we found that 2 and 7 of the SnocGRs and SnitGRs, respectively, are GRs for sugar taste, and most were found to be trehalose receptors.
In the phylogenetic tree ( Fig. 7) of GR sequences, there are two sugar taste lineages and two bitter taste lineages. One SnocGR and 3 SnitGRs clustered in the sugar taste lineages and no Sirex GRs clustered in the bitter taste lineages. Most Sirex GRs exhibited homology to sugar taste receptors, which may indicate that the function of GRs in the Sirex olfactory system is to detect carbohydrates.

. The expression values (FPKM) of 4 SnocIRs and 11
SnitIRs were greater than 1. Of these, SnocIR6 and SnitIR6 have the greatest expression values in the antennal transcriptome.
Previous studies have indicated that IR8a and IR25a are the co-receptors of IRs. In our phylogenetic tree (Fig. 8 pairs of NMDA receptors are found in the phylogenetic tree.

Homologous olfactory system genes in S. noctilio and S. nitobei
Among the olfactory genes identified in the transcriptome, we observed that some of the same types of olfactory genes are located on the same transcripts, such as SnocOBP10 and SnocOBP16, and SnitCSP3 and SnitCSP4. The distances between them are nearly 1,000 bp, which may explain their evolutionary relationship.
We used the olfactory genes of the two woodwasp species to build multiple phylogenetic trees (Fig. 9). In our phylogenetic tree, most of the S. noctilio and S. nitobei olfactory genes clustered together. Additionally, we found that most of the S. noctilio and S. nitobei olfactory genes are homologous, supporting the close evolutionary relationship between the two species.
Heatmap analysis demonstrates the differential expression between homologous genes of S. noctilio and S. nitobei, such as SnocOR1 and SnitOR1, SnocOR3 and SnitOR3, SnocOR5 and SnitOR5, SnocOR17 and SnitOR17, and SnocOR20 and SnitOR20. These differentially expressed genes may denote molecular mechanisms underlying the reproductive isolation between the two species. High expression of SnocOBP7 and SnitOBP7 was detected in male externalia, and SnocOBP3 was primarily expressed in the genitalia of female S. noctilio. SnocOBP10 was mainly expressed in male heads, while SnitOBP3 and SnitOBP10 did not show obvious tissue bias due to low expression levels.
Significant sex-biased expression was observed for many OBP genes, including SnocOBP3, SnocOBP4, SnocOBP7, SnocOBP10, SnocOBP15, SnitOBP4, SnitOBP7, and SnitOBP15. This sex bias may denote different functions of these OBPs between males and females, such as the perception of the opposite sex or oviposition behavior. We found that some homologous genes differ greatly in their expression profiles between the two species, especially those not expressed in the antennae. The differential expression of these homologous genes may indicate that they strengthened or lost their original function during species differentiation, resulting in olfactory differences between the two species.

Discussion
The number of OBPs varies greatly among different species Evolution of OBPs is mainly driven by lineage-specific amplification, with few distinct homologues in non-relative species [56][57][58] [62][63][64][65]. It is generally believed that more singular and closed environments are associated with less lineage-specific evolution of olfactory function.
Consequently, these insects have fewer olfactory genes as a result of positive selection pressure [64,66]. S. noctilio and S. nitobei live in pure forests or mixed coniferous forests and thus are more likely to receive plant volatiles or interspecies pheromone substances with a more concentrated population density, so the number of OBPs in Sirex spp. is less.

Special subfamilies of OBPs in Sirex
We did not uncover any Plus-C OBPs in the transcriptomes of S. noctilio and S. nitobei. Plus-C OBPs were found in Lepidoptera ( B. mori), Diptera (D. melanogaster), Coleoptera (Anoplophora chinensis), but were not found in existing Hymenoptera genomes or transcriptomes [67]. For example, Plus-C OBPs were not found in the genomes of Hymenoptera such as A. mellifera and N. vitripennis. This finding suggests that the Plus-C subtype is rare or even absent in Hymenoptera and has had a weak influence on the evolution of olfactory recognition in Hymenoptera.
Although the Plus-C subtype has not been discovered in Hymenoptera, the Minus-C subtype 19 An olfactory gene in the venom gland of S. noctilio In a previous report, four OBPs and five CSPs were found in the venom gland proteome of S.
noctilio [69]. Through sequence alignment, it was found that the OBPs identified in the venom gland were SnocOBP2, SnocOBP6, SnocOBP9, and SnocOBP11, and the CSP identified was SnocCSP2-5, indicating that these OBPs and CSPs may play special roles in transport or recognition of chemical signals in both antennae and venom glands. CSP5, which was detected in the venom gland, was not detected in our antennal transcriptomes.
Olfactory genes colocalized on the same transcript For various olfactory genes, we have found that two or more genes are colocalized on the same transcript. In previous studies, it was found that olfactory gene families expand through gene duplication and subsequent evolution [70]. The colocalization of olfactory genes on the same transcript indicates that these genes share a common ancestor gene and supports the gene duplication model of olfactory gene family expansion.

Characteristics and function of ORco and ORs with sex-biased expression
It has been shown that some ORs bind a single odor signal, while other ORs are able to respond to a low concentration of a single compound as well as higher concentrations of other substances [22]. ORs play an important role in the selectivity of the insect olfactory system, but other olfactory proteins may contribute to its overall specificity [22]. ORco has been observed in Diptera, Lepidoptera, Coleoptera, and Hymenoptera. Compared to traditional odor receptors, ORco is highly conserved, and its homology among insects can reach 50%-99%. Amino acid sequence analysis revealed a highly conserved region at the end of the ORco sequence [71].

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There are four OR genes ( SnocOR18, SnocOR30, SnitOR18, and SnitOR30) in the two Sirex species, and their expression levels in the antennae display 10-fold differences between the sexes. Some of these OR genes exhibit high male-biased expression, suggesting that these four OR genes may function as sex pheromone receptors. SnocOR30 and SnitOR30 display high homology with AmelOR170. AmelOR170 has shown a biased expression pattern in drone antennae, but the receptor does not bind 9-oxo-2-decenoic acid [72]. MmedOR9, which is homologous to SnocOR18 and SnitOR18, is also highly expressed specifically in males, suggesting that these genes may be pheromone receptors that interact with each other in Hymenoptera [73].
Expression of trehalose receptors in Sirex spp.
We identified 2 and 7 sweet receptors in the transcriptomes of S. noctilio and S. nitobei, respectively. Most of these sweet receptors were found to be trehalose receptors. Trehalose is a non-reducing sugar composed of two α-glucose molecules joined by an 1,1-glycosidic bond. The sugar is chemically stable and protects plants, plant cells, and plant proteins from freezing and drying. It is a stress-resistant protection mechanism. Trehalose is also present in the body fluid of insects and can be used as a energy source for flying. As an important blood sugar in insects, trehalose is present in almost all tissues and organs of insects [74]. Trehalose can influence insects' choice for food via recognition by GRs. Thus, trehalose receptors are important for vital biological processes in insects.
The expression pattern of OBPs in Sirex spp.
SnocOBP9 and SnitOBP9, which are PBP homologues, are mainly expressed in antennae and thus could be speculated to play a key role in identification of pheromone components. The weak sex bias and high levels of tissue expression may indicate that both males and 21 females need these OBPs to sense sex pheromones or aggregation pheromones.
SnocOBP11 and SnitOBP11 display significant sex-biased expression in the female external genitalia and male antennae. This may indicate that OBP11 can detect and control the number of sex pheromones released by females, and that male woodwasps receive sex pheromones through OBP11. This expression profile is distinct from OBPs of other insects, further indicating that OBP11 may be specialized proteins that receive sex pheromones in woodwasps.
A substantial number of OBPs exhibit a highly specific expression pattern in the male and female genitalia, which may indicate that the external genitalia can recognize sex pheromones or plant volatile components, and help guide normal behaviors such as feeding, mating, or spawning.

Assembly and function annotation
Trimming and quality control of the raw paired-end reads was performed using SeqPrep Jing-Zhen Wang: PhD candidate; major: forest protection; study direction: insect molecular biology and insect chemical ecology.

Competing interests
The authors declare that they have no competing interests.

Consent for publication
Not applicable.
Ethics approval and consent to participate The woodwasps, S. noctilio and S. nitobei (Hymenoptera: Siricidae) are forestry pests in China, which collections were made with the direct permission of Tongliao forestry bureau.
They are not included in the "List of Endangered and Protected Animals in China". All operations were performed according to ethical guidelines in order to minimize pain and discomfort to the insects.
39 Table S3. Sequence information and best blasts match information of odorant receptors (ORs). Table S4. Sequence information and best blasts match information of sensory neuron membrane proteins (SNMPs). Table S5. Sequence information and best blasts match information of gustatory receptors (GRs). Table S6. Sequence information and best blasts match information of ionotropic receptors   Candidate chemosensory proteins (CSPs) of Hymenoptera (purple), Diptera (green), and Lepidoptera (orange) are displayed in a neighbor-joining phylogenetic tree. SnocCSPs and SnitCSPs are marked with red and blue arrows, respectively.     Odorant binding protein (OBP) transcript levels with antennae-biased expression in 4 tissues of male and female Sirex. S. noctilio data is colored red and S. nitobei data is colored blue. The expression level of the male genitalia was set to 1 in order to obtain the relative expression in each tissue. The expression of SnitOBP15 in male antennae was set to 1 because the expression of male genitalia was too low to display.