Introduction
Insect behaviors, including mating, foraging, host-seeking, and oviposition are guided by their olfactory systems (Krieger and Breer, Reference Krieger and Breer1999). Antennae are the major insect olfactory organs, on which hair-like sensilla distributed over the surface and are utilized to detect chemical signals from the environment (Keil, Reference Keil1984a, Reference Keil1984b; Larsson et al., Reference Larsson, Hallberg, Kozlov, Francke, Hansson and Lofstedt2002). Odorant binding proteins (OBPs) (Vogt and Riddiford, Reference Vogt and Riddiford1981) and odorant receptors (ORs) (Clyne et al., Reference Clyne, Warr, Freeman, Lessing, Kim and Carlson1999; Vosshall et al., Reference Vosshall, Amrein, Morozov, Rzhetsky and Axel1999) have been reported to play pivotal roles in the dynamics, selectivity, and sensitivity of the insect olfactory system.
OBPs are one group of small proteins and highly expressed in olfactory tissues, which can bind, solubilize, and transport semiochemical molecules from the environment to the receptors (Leal, Reference Leal, Blomquist and Vogt2003; Pelosi et al., Reference Pelosi, Iovinella, Zhu, Wang and Dani2018). The first insect OBP was identified from Antheraea polyphemus at the beginning of 1980s by using the tritium labeled specific pheromone (E,Z)-6,11-hexadecadienyl acetate as a probe (Vogt and Riddiford, Reference Vogt and Riddiford1981). The functional roles of insect OBPs have been addressed by using in vivo technologies. For example, the deletion of a LUSH (OBP of the Drosophila melanogaster) gene suppresses D. melanogaster electrophysiological and behavioral response to the male pheromone 11-cis-vaccenyl acetate (Xu et al., Reference Xu, Atkinson, Jones and Smith2005). ORs are chemosensory receptors localized on the dendritic membrane inside the olfactory sensilla (Sato et al., Reference Sato, Pellegrino, Nakagawa, Vosshall and Touhara2008; Wicher et al., Reference Wicher, Schafer, Bauernfeind, Stensmyr, Heller, Heinemann and Hansson2008), detect volatile compounds, and transduce the olfactory signals to insect brain to regulate behavior. With the advances on the next-generation sequencing methods and the completion of the genome or transcriptome sequences of multiple insect species, more and more OBP and OR genes have been annotated and studied.
Diadegma semiclausum is a parasitoid wasp, which specifically lays its eggs into the developing larvae of the diamondback moth, Plutella xylostella (Bai et al., Reference Bai, Cai, Li and Chen2009). P. xylostella is a major agricultural pest which feed on cruciferous plant species such as canola, cabbage, cauliflower, and broccoli. It is one of the most destructive pests for Brassicaceae family crops and it can develop strong resistance to the common pesticides fast. D. semiclausum is the most commonly found parasitoid of P. xylostella in Australia, which is believed to provide a considerable aid in controlling P. xylostella. Diadegma spp. have been the most extensively studied and the broadly used biological control agents in the world. Biological control using parasitoid wasps such as D. semiclausum, became more and more important in integrated pest management strategies (Bukovinszky et al., Reference Bukovinszky, Gols, Posthumus, Vet and Van Lenteren2005). A previous study showed that P. xylostella infested mustard and Brussels sprout were more attractive to D. semiclausum than uninfested conspecifics (Bukovinszky et al., Reference Bukovinszky, Gols, Posthumus, Vet and Van Lenteren2005), suggesting that P. xylostella infested plants can release volatile compounds to attract D. semiclausum. However, limited attention has been paid to D. semiclausum olfactory system. The olfactory basis of D. semiclausum behaviors such as host-seeking and oviposition are still poorly investigated. The application of the next-generation sequencing provides an efficient and comprehensive approach to examine the olfactory genes and their expression profiles in parasitoid wasps and shed light into the molecular mechanism of their olfactory system. In this study, RNA sequencing of D. semiclausum antennae was performed to obtain the transcriptome database for identifying and investigating the key olfactory genes such as OBPs and ORs. Phylogenetic, bioinformatics, and molecular biology approaches were utilized to characterize these identified OBP and OR genes. The annotation and characterization of D. semiclausum OBPs and ORs will improve our understanding of its olfactory system, enhancing the identification of potential attractants using a reverse chemical ecology strategy (Leal et al., Reference Leal, Barbosa, Xu, Ishida, Syed, Latte, Chen, Morgan, Cornel and Furtado2008), and shed light on the functional investigations, which will assist in the development of more efficient and environmentally friendly pest control strategies. For example, using volatile compounds to attract D. semiclausum to the P. xylostella damaged crops at early stage for insect pest management in a more efficient and effective way.
Materials and methods
Insect, tissue collection, and RNA extraction
D. semiclausum pupae were sourced from Biological Services (https://biologicalservices.com.au/) and individually kept in a 7 ml screw top, clear vial (Sigma Aldrich, Australia) in the lab at 25 ± 1°C, 70–80% (R.H.) and 16:8 h (L:D) photoperiod. A 10% sugar solution was provided to feed emerging adults.
Based on the presence/absence of the ovipositor, male and female adults were differentiated under a stereo microscope. A total of 100 male and 100 female adults of 0–3 days old were collected and put to anesthesia using carbon dioxide (purity >99.9%) for 5 min. Then antennae were excised with a surgical scalped blade under a stereo microscope. All collected antennae were immediately stored in liquid nitrogen, from which total RNA was extracted using a RNeasy mini kit (Qiagen, USA) according to the manufacturer's protocol. The extracted total RNA from adult antennae was treated by DNase I to remove genomic DNA, quantified, and quality controlled using NanoDrop ND-2000 (Thermo Scientific, USA) and stored in a −80°C freezer in Western Australia State Agricultural and Biotechnology Centre (SABC). The extracted total RNA samples were sent to Beijing Genomics Institute (BGI) Hong Kong (https://www.bgi.com/us/) in dry ice quality control, generating Paired-End Illumina TruSeq libraries and sequencing with an Illumina HiSeq 2500/4000 platform. The RNA integrity number (RIN) values were 8.4 for male antennae RNA sample and 9.4 for female antennae RNA sample.
Sequence assembly
The raw data received from BGI were pooled for the transcriptome assembly. All the raw RNA-Seq reads were analyzed using FastQC (Andrews et al., Reference Andrews, Krueger, Seconds-Pichon, Biggins and Wingett2014) to check for read quality. Overall, the raw reads displayed reasonable quality. The pooled reads were assembled as pair-ended reads using Trinity, version 2.4.0 (Grabherr et al., Reference Grabherr, Haas, Yassour, Levin, Thompson, Amit, Adiconis, Fan, Raychowdhury and Zeng2011). Minimum contigs length was set to 200 bp. The contigs were then annotated using the Swissprot protein database.
Gene annotation and analysis
Genes encoding OBPs and ORs in D. semiclausum were identified using BLAST searches from the assembled transcriptome database with reported D. melanogaster and Apis mellifera OBPs and ORs as query. Extensive manual curation was then performed on the D. semiclausum OBP and OR genes. The identified OBP and OR amino acid sequences were used for validation by NCBI blast based on the identity and similarity to orthologous genes from other insects. All identified D. semiclausum OBP and OR amino acid sequences are available in an online supporting FASTA text file (Supplementary data and table 1). N-terminal signal peptides of DsemOBPs were predicted by using SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP). The calculated molecular weights (MW) and isoelectric points (pIs) were obtained by using ExPASy proteomics server (http://www.expasy.org/tools/protparam.html) on the mature proteins (without signal peptides). The amino acid sequences of DsemOBPs were used to search the best blast hit sequences from NCBI by using blastp (table 1).
Table 1. D. semiclausum OBPs
Phylogenetic analysis
Encoded DsemOBP proteins were aligned using default settings for ClustalW in Geneious alignment (Geneious Prime). Gap Opening Penalty (10.00) and Gap Extension Penalty (0.10) were used for multiple sequence clustal alignment with 30% delay divergent cutoff.
The DsemOBP and DsemOR amino acid sequences were used to create entry file for phylogenetic analysis in Geneious with other OBP, and OR protein sequences from P. xylostella or other Hymenoptera species including A. mellifera, Trichogramma pretiosum, Nasonia vitripennis and Diachasma alloeum (Robertson and Wanner, Reference Robertson and Wanner2006; Robertson et al., Reference Robertson, Gadau and Wanner2010; Tvedte et al., Reference Tvedte, Walden, McElroy, Werren, Forbes, Hood, Logsdon, Feder and Robertson2019). A. mellifera is one of the model species for Hymenoptera, whose olfaction and social behaviors have been extensively studied. N. vitripennis is the most widely studied of the parasitoid wasps, which is a generalist and parasitizes a wide range of dipteran hosts, including blowflies, fleshflies, and houseflies. D. alloeum is a specialist parasitoid of the fruit fly, Rhagoletis pomonella. OBPs and ORs from these three species have been annotated and published (Robertson and Wanner, Reference Robertson and Wanner2006; Robertson et al., Reference Robertson, Gadau and Wanner2010; Tvedte et al., Reference Tvedte, Walden, McElroy, Werren, Forbes, Hood, Logsdon, Feder and Robertson2019). Both T. pretiosum and D. semiclausum are parasitoid wasps of P. xylostella but the former is an important natural egg parasitoid and it can lay eggs into Helicovpera and Spodoptera eggs as well. In another project conducted by us, 22 OBPs and 105 ORs were identified from T. pretiosum genome (unpublished data). First, the amino acid sequences of OBPs and ORs were aligned using Geneious alignment with default settings: global alignment with free end gaps, cost matrix (Blosum62), gap open penalty (12), gap extension penalty (3) and refinement iterations (2). A Geneious tree was then constructed using default settings based on Jukes–Cantor model with bootstrap as the resampling method, and 1000 replicates and 50% support threshold. The same method was used to study the OBP and OR genes between D. semiclausum and P. xylostella in order to give an insight into the interactions between the parasitoid and its host insects.
Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time (qRT-PCR)
Total RNA from D. semiclausum male and female adult antennae was extracted using the Qiagen RNeasy mini kit (Qiagen, USA), treated by DNase I (New England Biolabs, USA) to remove genomic DNA, quantified and quality checked using NanoDrop™ ND-2000 (Thermo Scientific, USA) as described above. The cDNA templates were prepared from purified RNA samples using the SuperScript® VILO™ cDNA synthesis kit (Invitrogen, USA), according to the manufacturer's manual. RT-PCR was performed using gene-specific primers as shown in table 2, which were designed by using http://bioinfo.ut.ee/primer3/. RT-PCR was performed using Taq DNA Polymerase (New England Biolabs, Australia) as follows: 95°C for 30 s; 41 cycles at 95°C for 25 s, 55°C for 30 s and 68°C for 20 s; and final extension at 68°C for 5 min then hold at 10°C. The PCR products were visualized using 1.0% agarose gel electrophoresis under 75 V supplied by a Bio-Rad 1000/500 power supply (Xu et al., Reference Xu, Liu, Liao and Anderson2017). The agarose gels were examined under the Bio-Vision system (Vilber Lourmat, France).
Table 2. DsemOBP primers designed for RT-PCR and qRT-PCR
qRT-PCR was performed using the same gene-specific primers as in table 2 and reference genes included were DsemRPL8, DsemRPL13a and DsemORco. A two-step qPCR protocol was performed on Rotor-Gene Q-5 Plex (Qiagen, USA) using Power SYBR® Green PCR Master mix (Thermo Fisher Scientific, Australia) following the protocol Bio-Rad CFX96 Real-Time PCR Detection System (Xu and Liao, Reference Xu and Liao2017). For each cDNA sample and primer set, reactions were run in triplicate, and average fluorescence Ct values were obtained. Relative expression levels were determined using the comparative 2−ΔΔCt method for relative quantification (Livak and Schmittgen, Reference Livak and Schmittgen2001). Statistical analysis was performed on the expression profiles between male and female adults using the t-test in SPSS. The symbol * means P value <0.05, ** means P value <0.01 while *** means P value <0.001.
In silico expression profiles of DsemORs
Using STAR aligner version 2.5.3a (Dobin et al., Reference Dobin, Davis, Schlesinger, Drenkow, Zaleski, Jha, Batut, Chaisson and Gingeras2013), the retained reads were independently aligned against the assembled transcriptome library. Alignment parameters were set such that a maximum of two mismatches were allowed. Mismatches of two were used to accommodate potential base-calling errors. Only uniquely aligned reads were retained for analysis while reads aligned to multiple loci were discarded. A count matrix was generated and loaded into R (version 3.5.1) for downstream statistical analysis. The transcripts were removed from analysis if less than ten unique reads could be aligned. Fragments per kilobase million values were used as the main criterion to identify differentially expressed (D.E.) genes (Xu and Anderson, Reference Xu and Anderson2015).
Results
Identification of DsemOBPs
The transcriptome assembly of male and female D. semiclausum antennae consists of 84,668 contigs and has a total size of 123,007,582 bp, with an N50 of 2742 bp. A total of 33,295 transcripts were estimated. The maximum contig is 16,148 bp and the average size is 1452 bp. In total, 24,660 transcripts passed the quality control criteria and were evaluated for differential expression analysis.
Here, 17 DsemOBPs were identified from D. semiclausum transcriptome sequences and 11 of them are full-length sequences, including DsemOBP6-13 and DsemOBP15-17. All the others are only partial sequences either missing the N-terminal or C-terminal (table 1). In these 11 full-length DsemOBPs, no signal peptide was predicted from DsemOBP16 and DsemOBP17. The mature (without signal peptide) DsemOBPs with full length range from 108 to 231 amino acids and their molecular weights range from 11,926 to 27,093 Da (table 1). The pIs of most full-length DsemOBPs are below 7.0 but that of DsemOBP8 is above (pI: 8.25).
The newly identified DsemOBPs were aligned by using Geneious clustal alignment. The alignment of these DsemOBP amino acid sequences highlights the six conserved cysteine residues (fig. 1). Most of the D. semiclausum OBPs share the characteristic features of the classic OBP family: small size, presence of an N-terminal signal peptide sequence as well as a highly conserved pattern of six cysteine residues called the classic motif (fig. 1).
Figure 1. Alignment of 17 DsemOBP amino acid sequences. The six conserved cysteines were highlighted.
Phylogenetic analysis of DsemOBPs
The phylogenetic analysis on OBPs was performed using D. semiclausum, A. mellifera, T. pretiosum, N. vitripennis and D. alloeum OBP protein sequences (Robertson and Wanner, Reference Robertson and Wanner2006; Robertson et al., Reference Robertson, Gadau and Wanner2010; Tvedte et al., Reference Tvedte, Walden, McElroy, Werren, Forbes, Hood, Logsdon, Feder and Robertson2019). The results revealed that multiple species-specific OBP subfamilies were observed (fig. 2a) but no D. semiclausum-specific OBP subgroup was identified. Various DsemOBPs were clustered with OBPs from other Hymenoptera species (fig. 2a). DsemOBP5, DsemOBP6, and DsemOBP7 were clustered on the same branch with TpreOBP8, suggesting these three DsemOBPs may play similar roles to TpreOBP8. For example, detecting P. xylostella, the same host insect. They may detect plant compounds for nectar or sugar feeding as well, which provide insects with the critical energy resources, or detecting P. xylostella to lay eggs. Similarly, DsemOBP13 was clustered with DallOBP11, DsemOBP14 was clustered with NvitOBP70, and DsemOBP4 was clustered with NvitOBP42, suggesting these DsemOBPs may play similar functions in related insect species.
Figure 2. Phylogenetic trees of DsemOBPs. (a) Phylogenetic analysis of OBPs from D. semiclausum (DsemOBPs), T. pretiosum (TpreOBPs), A. mellifera (AmelOBPs), N. vitripennis (NvitOBPs) and D. alloeum (DallOBPs). Seventeen DsemOBPs were marked in red, 22 TpreOBPs were marked in green, 21 AmelOBPs were marked in blue, 90 NvitOBPs were marked in black and 15 DallOBPs were marked in orange. (b) The phylogenetic analysis of DsemOBPs and PxylOBPs. Seventeen DsemOBPs were marked in red while 39 PxylOBPs were marked in black. Consensus support values (%) were labeled on branches.
Furthermore, to study the similarities of OBPs between the host insect (P. xylostella) and the parasitoid wasp (D. semiclausum), the phylogenetic analysis was performed between 17 DsemOBPs and 39 PxylOBPs (Cai et al., Reference Cai, Zheng, Huang, Xu and You2021). The results showed that most of OBPs are species-specific. There is no DsemOBP clustered with P. xylostella PBP/GOBP subfamily (fig. 2b), a conserved OBP group in Lepidopera species (Cai et al., Reference Cai, Cheng, Qin, Xu and You2020). Interestingly, DsemOBP13 and PxylOBP23 were clustered together, which shared 26.52% identities at the amino acid level. DsemOBP5 and PxylOBP31 were clustered together, which showed 35.25% identity at the amino acid level.
Expression profiles of D. semiclausum OBPs
Expression profiles of DsemOBPs can help build the links between the genes and their functions. Here, 17 identified DsemOBPs were examined between male and female adult antennae using RT-PCR. All 17 DsemOBPs were successfully amplified and the band sizes were the same as expected in the RT-PCR analysis (fig. 3), suggesting the primers were designed properly. DsemOBP2 and DsemOBP5 were only amplified from male antennae, suggesting that they may play male-specific roles, for example, detecting sex pheromones released by female or localizing the females for mating. Other DsemOBPs were amplified in both male and female adult antennae, which may play the same roles between male and female adults such as detecting plant volatile compounds and assist the wasps to localize nectar as energy resource.
Figure 3. RT-PCR analysis of 17 DsemOBPs between adult male and female adults. M, male and F, female.
To study the expression levels of DsemOBPs, the qRT-PCR approach was applied using D. semiclausum RPL8 and RPL13a genes as reference genes (fig. 4). The results showed that there are clearly three types of expression levels of DsemOBPs. Type 1, male-specific or male-rich OBPs, which means the OBP expression level was significantly higher in male antennae than that in female antennae, including DsemOBP 1, 3, 4, 5, and 16 (fig. 4). Type 2 are the female-specific or female-rich OBPs, which were significantly highly expressed in female antennae compared to male antennae, including DsemOBP 6, 7, 8, 9, 10, and 14. In these female-specific/rich OBPs, DsemOBP6 and DsemOBP7 share 87.5% identity, while DsemOBP10 and DsemOBP14 share 40.5% identity at the amino acid level. The other DsemOBPs which did not show significant differences in the expression levels between male and female belong to type 3 (fig. 4).
Figure 4. Relative expression profiles of 17 DsemOBPs by using quantitative RT-PCR. DsemRPL8 and DsemRPL13a genes were used as the reference genes for the normalization. Error bars show standard deviation. M, adult males and F, adult females. *P < 0.05, **P < 0.01, ***P < 0.001.
Furthermore, another reference gene, DsemOrco, was used in this study to normalize the expression profiles of DsemOBPs between male and female antennae. DsemRPL9 and DsemRPL13a are house-keeping genes which can be expressed through the whole body. DsemOrco is an antennae specific co-receptor gene, which assists other ORs to function appropriately in the antennae. Therefore, it is believed that Orco is a more suitable reference gene to normalize the expression of the olfactory genes in the antennae (Pelletier and Leal, Reference Pelletier and Leal2009). The results (Supplementary fig. 1) are quite similar as using RPL8 and RPL13a (fig. 4). DsemOBP6, 7, 8, 9, 10, 11 and 14 showed significantly higher expression in female antennae than in male antennae, which may play important roles for female specific behaviors such as host-seeking and oviposition on P. xylostella. In the qRT-PCR analysis using DsemRPL9 and DsemRPL13a as reference genes, DsemOBP11 showed no significant differences in the expression levels between male and female adults (fig. 4). However, here DesemOBP11 exhibited higher expression in female than male adults when DsemOrco was used as the reference gene (Supplementary fig. 1). Similarly, DsemOBP1 and DsemOBP16 exhibited higher expression in male than in female in the qRT-PCR analysis using DsemRPL9 and DsemRPL13a as reference genes but showed no significant differences between male and female when using DsemOrco as the reference gene (Supplementary fig. 1).
Phylogenetic analysis and expression profiles of DsemORs
A total of 67 OR genes were identified from D. semiclausum, including DsemOrco gene. The phylogenetic analysis between D. semiclausum and T. pretiosum ORs showed that most ORs are species-specific (fig. 5a). However, a few TpreORs and DsemORs were clustered together in the phylogenetic tree. For example, TpreOR39 and DsemOR30, TpreOR33 and DsemOR19, TpreOR77 and DsemOR63, suggesting they may play the same roles between these two species. For example, detecting P. xylostella related odorants for oviposition or flower odorants for nectar sucking.
Figure 5. Phylogenetic analysis and expression profiles of DsemORs. (a) The phylogenetic analysis of 67 DsemORs and 105 TpreORs. 67 DsemORs were marked in red while 105 TpreORs were marked in black. (b) The phylogenetic analysis of DsemORs and PxylORs. Sixty-seven DsemORs were marked in red while 95 PxylORs were marked in black. Consensus support values (%) were labeled on branches and scale bar was showed. (c) The in-silico expression profile of DsemORs in male and female antennae (dark red, max. value; yellow, mid. value; and white, min. value).
Further, the phylogenetic analysis of the ORs from D. semiclausum and its host P. xylostella was conducted (fig. 5b). Both two insects live in the same environment, so they are very likely to detect the same compounds that regulate their behaviors. However, the results showed that no ORs are conserved between these two species except Orco.
Using transcriptome sequencing data, a heat map of the expression profile of the 67 DsemORs between male and female antennae was built (fig. 5c). A number of OR genes showed male antennae specific or rich expression, for example, DsemOR1, 4, 5, 6, and 49. They may play important roles in the reception of the sex pheromone and mating behaviors. Various OR genes showed female antennae specific or rich expression, including DsemOR2, 12, 16, 32, and 35, which may function for the female adults to detect the P. xylostella larvae for oviposition.
Discussion
In this study, 17 OBP and 67 OR genes were identified from D. semiclausum transcriptome sequencing, which were further compared to OBPs and ORs from P. xylostella, the host insect, and other Hymenoptera species including A. mellifera, T. pretiosum, N. vitripennis, and D. alloeum (Robertson and Wanner, Reference Robertson and Wanner2006; Robertson et al., Reference Robertson, Gadau and Wanner2010; Tvedte et al., Reference Tvedte, Walden, McElroy, Werren, Forbes, Hood, Logsdon, Feder and Robertson2019). The expression patterns of D. semiclausum OBPs between male and female antennae were examined using RT-PCR and qRT-PCR. Six OBPs (DsemOBP 6, 7, 8, 9, 10, and 14) demonstrated significantly higher expression in females than in males, which may assist in female D. semiclausum host-seeking and oviposition behaviors. DsemOR2, 12, 16, 32, and 35 showed female antennae specific/rich expression, indicating they may function for the female adults to detect the P. xylostella larvae for oviposition.
Insects and their natural enemies co-exist in the same ecosystem, so they can use the semiochemicals around them to help detect each other and regulate their behaviors, for example, prey and flee. Many plants attacked by herbivore insects can emit leaf volatile organic compounds that attract the herbivore insects’ natural enemies, such as parasitoids and predators (Dicke et al., Reference Dicke, Sabelis, Takabayashi, Bruin and Posthumus1990) to control the pests. One example is that aphids can secrete droplets of sticky fluid in an attempt to keep parasitoids and predators away, which contains an alarm pheromone, (E)-β-farnesene (Bowers et al., Reference Bowers, Nault, Webb and Dutky1972; Wientjens et al., Reference Wientjens, Lakwijk and van der Marel1973). Alarm pheromone may act as primer or releaser thus eliciting conspecific physiological and behavioral responses, respectively. Parasitoids and predators, on the other hand, eavesdrop on aphid communication and utilize (E)-β-farnesene as a kairomone, enhancing foraging behaviors (Hatano et al., Reference Hatano, Kunert, Michaud and Weisser2008). All these studies demonstrated that insects and natural enemies can detect certain same odorant compounds by using their olfactory systems. These detections indicate they may possibly share certain conserved olfactory proteins like OBPs or ORs to help this process. It was reported that aphids and their predators share highly conserved OBPs. Purified recombinant OBPs from the English grain aphid, SaveOBP3, and the marmalade hoverfly, EbalOBP3, specifically bind (E)-β-farnesene with apparent high affinity (Vandermoten et al., Reference Vandermoten, Francis, Haubruge and Leal2011). However, a following correction research (https://doi.org/10.1371/annotation/6fb8a803-8203-429c-b0d1-4d0d995c39e9) concluded that the orthologs from lady beetle (HaxyOBP3) and the hoverfly (EbalOBP3) are artifacts, probably derived from the template from the European grain aphid, Sitobion avenae. Whether there are conserved OBPs or ORs shared between insects and their natural enemies is still unknown. In our previous study, a number of compounds that elicit physiological or behavioral responses of P. xylostella, can also initiate D. semiclausum electroantennography (EAG) responses (unpublished data). For example, cis-11-hexadecanal is a sex pheromone component of P. xylostella. D. semiclausum showed high antennae responses to this compound in EAG analysis, suggesting that D. semiclausum may have a receptor for cis-11-hexadecanal to help D. semiclausum to locate the P. xylostella for parasitism (unpublished data). Therefore, D. semiclausum and P. xylostella are sound models for a comparative study between their OBPs and ORs. To perform an insightful study on these OBP and OR genes, the first step is to identify these genes. Moreover, a previous study showed that pest-infested plants were more attractive to parasitoid wasps than uninfested conspecifics (Bukovinszky et al., Reference Bukovinszky, Gols, Posthumus, Vet and Van Lenteren2005), indicating that plants may release volatile compounds to attract wasps after they were attacked. For example, plants can effectively attract wasps, Opius dissitus, by releasing a universally induced compound, (Z)-3-hexenol, and potentially protect these plants safe from parasitic assaults by leafminer pests, Liriomyza huidobrensis (Wei et al., Reference Wei, Wang, Zhu, Zhang, Nandi and Kang2007). The identification and characterization of the OBP and OR genes from D. semiclausum may help further identifying these volatile compounds released from P. xylostella infested host plants using the reverse chemical ecology approach (Leal et al., Reference Leal, Barbosa, Xu, Ishida, Syed, Latte, Chen, Morgan, Cornel and Furtado2008), which are candidate attractants to D. semiclausum.
The numbers of ORs in Hymenoptera species are generally very high. For example, 177 OR genes have been identified from European honeybee (A. mellifera) and such large number of ORs may be used to detect the odorants from various flowers for nectars and pollens. It is not surprising that a significantly lower number of OR genes (only 67) were identified from D. semiclausum because here only a transcriptome data on the antennae was established, not a genome sequence. Many OR genes with low expression in the antennae may not be identified by using transcriptome sequencing. Moreover, OR genes that are specifically expressed in other olfactory tissues including, larval antennae and adult palps, as reported in other insect species (Liu et al., Reference Liu, Xu, Papanicolaou, Dong and Anderson2014), were not identified yet. Once the genome sequencing of D. semiclausum is available, more ORs will be identified. Moreover, D. semiclausum is a parasitoid wasp and spend immature (egg and larvae) stages in their host insects (P. xylostella), which may decrease the number of ORs in the evolution since they do not need a robotic and sensitive olfactory system at the larvae stage.
Then, the D. semiclausum OBP and OR genes were compared with P. xylostella OBP and OR genes to investigate if they share certain similar olfactory genes. P. xylostella OBP and OR have been annotated and published (You et al., Reference You, Yue, He, Yang, Yang, Xie, Zhan, Baxter, Vasseur, Gurr, Douglas, Bai, Wang, Cui, Huang, Li, Zhou, Wu, Chen, Liu, Wang, Li, Xu, Lu, Hu, Davey, Smith, Chen, Xia, Tang, Ke, Zheng, Hu, Song, You, Ma, Peng, Zheng, Liang, Chen, Yu, Zhang, Liu, Li, Fang, Li, Zhou, Luo, Gou, Wang, Wang, Yang and Wang2013; Cai et al., Reference Cai, Zheng, Huang, Xu and You2021), which provide an important database for this study. Insect hosts and their parasitoid wasps, which always dwell in the same ecosystem, may share similar OBPs or ORs to help detect same volatile compounds from their environment. The results showed that certain DsemOBPs and PxylOBPs exhibit similarities but not ORs. D. semiclausum and P. xylostella are two totally different species: one is Hymenopteran and the other is Lepidopteran. No conserved OR genes between these two species were identified. DsemOBP13 and PxylOBP23 shared 26.52% identity at the amino acid level. DsemOBP5 and PxylOBP31 showed 35.25% identity. These DsemOBPs are the target proteins for further examination to answer the questions such as: Which compounds do they bind? Are they the same compounds? Will a knock-down or a knock-out of these genes, disrupt D. semiclausum parasitic behavior on P. xylostella?
No sequence similarity does not mean those ORs cannot bind or detect same compounds. For example, an endogenous OR in the fruit fly, D. melanogaster, is highly sensitive to the sex pheromone of the silkworm moth, bombykol (Syed et al., Reference Syed, Kopp, Kimbrell and Leal2010). Intriguingly, the fruit fly detectors are more sensitive than the receptors of the silkworm moth, although its ecological significance is unknown. These two receptors showed extremely low similarities at the amino acid level.
Only female D. semiclausum adults lay eggs on the host insect eggs, so the female-specific/rich OBPs or ORs are more likely to play a significant role in the parasite behaviors. DsemOBP6-10, 14, and DsemOR2, 16, 32, and 35 are female-specific or female-rich, which are the candidate OBPs and ORs to assist in D. semiclausum host-seeking and oviposition behaviors. Male-specific/rich OBPs and ORs were also identified including DsemOBP 1, 3, 4, 5, and 16 and DsemOR4-6, 20, 21, and 49, suggesting they may play a role in assisting male-specific behaviors, for example, following chemical signals (e.g., sex pheromone) to find females for mating.
Furthermore, T. pretiosum and D. semiclausum are both important parasitoid wasps, which are widely used in the biological control for P. xylostella. However, T. pretiosum is a natural egg parasitoid and can lay eggs to host insect eggs including P. xylostella. D. semiclausum lay eggs into P. xylostella larvae specifically and selectively. A comparative study on the olfactory systems of these two species will improve our understanding of their olfaction-related behaviors and biology. Various similar OBPs and ORs were identified between T. pretiosum and D. semiclausum (e.g., TpreOR39 and DsemOR30, TpreOR33 and DsemOR19, TpreOR77 and DsemOR63, TpreOBP2 and DsemOBP8), which needs further investigations to understand their functions. Here T. pretiosum genome sequence was available but D. semiclausum genome has not yet. Therefore, more conserved OBPs or ORs may be identified after D. semiclausum genome was published.
Conclusion
In summary, this study provides a first investigation of the olfactory genes of D. semiclausum at the molecular level. A number of candidate female-specific OBPs and ORs were identified including DsemOBP6-10, DsemOBP14, DsemOR2, DsemOR16, DsemOR32 and DsemOR35, which may play critical roles in D. semiclausum host-seeking and oviposition behaviors. DsemOBP13 and DsemOBP5 showed high identities to P. xylostella OBPs, suggesting they may help D. semiclausum detect similar compounds that P. xylostella can detect, which may help guide D. semiclausum to localize hosts through the semiochemical signals. Further investigations require functional studies of the D.E. genes we identified here to validate their roles in oviposition and host-seeking. Also, transcriptome analysis of other chemosensory tissues including maxillary palps, tarsi, or ovipositor may help identifying other chemosensory genes in this species and build links to their potential functions.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0007485321000675
Acknowledgements
We thank the Western Australian State Agricultural Biotechnology Centre (SABC) and the College of Science, Health, Engineering and Education (SHEE) at Murdoch University for their help with this project. Dr Wei Xu is the recipient of an Australian Research Council Discovery Early Career Researcher Award (DECRA) (DE160100382). Basman Al-Jalely is a recipient of a PhD. Scholarship was provided from the Iraqi Government, Ministry of Higher Education and Scientific research.
Conflict of interest
The authors declare that they have no competing interests.
