Temporal and Spatial Expression of Parasitism-Related Olfactory Genes in Larvae of the Ectoparasitoid Dastarcus helophoroides (Coleoptera: Bothrideridae)
Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) is an ectoparasitoid of the pine sawyer beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae). A sensitive and precise olfactory system is required for the accurate location of the coleopteran host by D. helophoroides neonates. Herein, we characterized the relative expression patterns of six representative olfactory-related genes at different stages of M. alternatus parasitism and in different body regions of D. helophoroides neonates. The genes encoding chemosensory protein 2 (DhelCSP2), odorant receptor 2 (DhelOR2), and ionotropic receptor 2 (DhelIR2) were significantly upregulated before parasitization was initiated, whereas the genes encoding odorant binding protein 8 (DhelOBP8), gustatory receptor 5 (DhelGR5), and sensory neuron membrane protein 1 (DhelSNMP1) were significantly upregulated 4–5 d after initiation of parasitism. In D. helophoroides neonates, four genes (DhelOBP8, DhelCSP2, DhelOR2, and DhelIR2) were significantly upregulated in the head compared with the thoracoabdominal region, and one gene (DhelGR5) was significantly upregulated in the thoracoabdominal area compared with the head. Double-stranded RNAs (dsRNAs) targeting the six olfactory-related genes were synthesized and delivered to D. helophoroides neonates via immersion. After dsRNA treatment, the transcript levels of four olfactory-related genes (DhelOBP8, DhelCSP2, DhelOR2, and DhelSNMP1) were significantly reduced compared with that of the controls. These results provide a basis for further functional explorations of D. helophoroides olfactory genes, which may lead to the development of improved biological pest control methods using D. helophoroides larvae.Abstract
Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) is an ectoparasitoid of the pine sawyer beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae), the primary insect vector of the pine wood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle (Aphelenchida: Parasitaphelenchidae) (Mamiya and Enda 1972, Taketsune 1982). In China, large-scale releases of laboratory-reared D. helophoroides adults and eggs have been used to reduce the population density of M. alternatus and inhibit the proliferation of B. xylophilus (Yang et al. 2014). In field experiments, D. helophoroides larvae effectively parasitized M. alternatus larvae from a distance of 7.5 m (Li et al. 2013). Therefore, we hypothesized that the keen olfactory system of the D. helophoroides larva plays an essential role in the parasitic process.
Insect behavioral processes, such as host search, foraging, pairing, and egg laying, require the olfactory system (Becher et al. 2012, Birkett et al. 2004, Carey et al. 2010, Leal 2013, Syed and Leal 2009, Todd et al. 1992). For example, the odorant binding protein (OBP) DhelOBP21 was shown to play a vital role in the recognition of (þ)-β-pinene in adult D. helophoroides (Li 2019). However, it is larval D. helophoroides that exhibit parasitic behavior; thus, the role of olfactory genes in the host search and parasitism of D. helophoroides neonates requires investigation.
To address this knowledge gap, we previously published the transcriptome of D. helophoroides neonates and characterized the expression patterns of 62 olfactory genes encoding six types of proteins: 20 encoding putative OBPs, 11 encoding putative chemosensory proteins (CSPs), 9 encoding putative odorant receptors (ORs), 11 encoding putative ionotropic receptors (IRs), 7 encoding putative gustatory receptors (GRs), and 4 encoding putative sensory neuron membrane proteins (SNMPs) (Li et al. 2020). To continue this investigation and to clarify the functions of the olfactory genes during that parasitism process, we aimed to determine the spatiotemporal specificity of parasitism-related olfactory genes by profiling olfactory gene expression levels in D. helophoroides larvae at different parasitic stages and in various body regions. Furthermore, we investigated the silencing efficiency of double-stranded RNA (dsRNA) with respect to the olfactory genes by targeting representative olfactory genes in D. helophoroides larvae in vitro by using RNA interference (RNAi). Our overall objective was to provide a theoretical basis for further exploration of the molecular mechanisms underlying the parasitism of M. alternatus by D. helophoroides larvae.
Materials and Methods
Insect and tissue collection. Adult D. helophoroides were reared in our laboratory, and females laid eggs on vellum tied to pine wood blocks. We incubated the vellum containing the eggs at 25°C and 50% relative humidity (RH), monitoring the eggs every 24 h and retrieving neonates with a small brush.
Larvae of the M. alternatus host were obtained from the xylem of Pinus massoniana Lambert trees in the Miaoshou Forest Farm (Jingde County, Anhui Province, China) and kept in separate centrifuge tubes with air-permeable holes at 8°C and 50% RH.
Using sterile forceps, we separated the heads and thoracoabdominal parts of 100 D. helophoroides neonates, placed the heads and thoracoabdominal parts in separate 1.5-ml centrifuge tubes, and stored the tubes at –80°C. We performed this procedure three times (a total of 300 larvae), creating three biological replicates.
Parasitism was initiated by placing five D. helophoroides neonates on the surface of each M. alternatus larva by using a small brush (day 0). We collected 20 D. helophoroides larvae from each M. alternatus larva on days 2–6 after parasitism initiation; the collected larvae were placed in 1.5-ml centrifuge tubes and stored at –80°C. We performed this procedure three times, collecting a total of 60 D. helophoroides larvae at each stage of parasitism.
cDNA library preparation. Total RNA was extracted from D. helophoroides neonates. The larvae were collected at different stages of parasitism, and the larval heads and thoracoabdominal parts were analyzed using EasyPure RNA kits (TransGen, Beijing, China) following the manufacturer's instructions. RNA integrity was determined using 1% agarose gel electrophoresis, and RNA concentration was measured using a Multiskan GO microplate spectrophotometer with a µDrop plate (Thermo Fisher Scientific, Waltham, MA). RNA was reverse transcribed to obtain complementary DNA (cDNA) by using an EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen) following the manufacturer's instructions.
Target gene selection and primer synthesis. In a previous study, we identified 62 olfactory-related genes encoding six types of proteins in the transcriptomes of D. helophoroides neonates (Li et al. 2020). From the genes identified in this previous study as strongly upregulated in D. helophoroides neonates, we selected one gene in each category for further investigation: DhelOBP8, DhelCSP2, DhelOR2, DhelIR2, DhelGR5, and DhelSNMP1. Primers for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were designed based on the previously published gene sequences using Primer3web (Version 4.1.0) (Table 1).

dsRNA synthesis. Using the TransStart KD Plus PCR SuperMix kit (TransGen), we amplified the target olfactory genes (DhelOBP8, DhelCSP2, DhelOR2, DhelIR2, DhelGR5, and DhelSNMP1) from the D. helophoroides larval cDNA and the green fluorescent protein (EGPF) gene fragment (as the control) from the PEGFP-C1 plasmid (Miaolingbio, Wuhan, China). PCRs were performed using a PCR instrument (Langji, Hangzhou, China), and primers were designed using Primer3-web (Version 4.1.0) (Table 2). All primers were supplemented with T7 promoter sequences to produce double-stranded DNA. PCR amplicon integrity was confirmed on 1% agarose gels before purification with SanPrep Column DNA Gel Extraction kits (Sangon, Shanghai, China). The purified products were sequenced by Sangon by using a 3730XL DNA Analyzer (Applied Biosystems, Foster, CA) to guarantee sequence accuracy and then used as templates for the synthesis of dsRNA with the MEGAscript T7 Transcription kit (Thermo Fisher Scientific).

dsRNA delivery. Using a small brush, 100 D. helophoroides neonates were placed in the center of a clean slide, and 10-µl drops of synthesized dsRNA (500 ng/ µl) were aspirated onto the gathered larvae with a pipette. After 10 min, the dsRNA solution was absorbed with filter paper and the larvae were washed twice with sterile water. The treated larvae were transferred to 1.5-ml of air-permeable centrifuge tubes by using a small brush and incubated at 25°C for 24 h. Three independent biological replicates of the dsRNA inhibition experiment were performed for each target olfactory gene and EGFP (the control). After total RNA was extracted from the treated larvae and reverse transcribed into cDNA as described previously, RNAi-mediated knockdown efficiency was determined using RT-qPCR.
RT-qPCR. The six olfactory genes and EGFP (control) were amplified from the cDNA templates by using PerfectStart Green qPCR SuperMix (TransGen) following the manufacturer's instructions. RT-qPCRs were performed on a LineGene 4800 Fluorescent Quantitative PCR Detection System (Bioer, Hangzhou, China). The thermal cycling conditions were 94°C for 30 s, followed by 40 cycles of 94°C for 5 s and 60°C for 30 s. Actin was used as the internal reference gene, and relative mRNA expression levels were calculated using the 2–ΔΔCT method (Livak and Schmittgen 2001).
Statistical analysis. Significant differences in the relative expression profiles of olfactory genes between D. helophoroides neonates and larvae at various stages of parasitism were identified using one-way analyses of variance and Tukey's tests. We also used Student's paired-samples t tests to compare transcript levels between larval heads and thoracoabdominal parts as well as between treatment and control groups in the RNAi experiments. All statistical analyses were performed using IBM SPSS Statistics for Windows, Version 26.0 (IBM Corp., Armonk, NY).
Results
Relative expression of olfactory genes in D. helophoroides larvae at different stages of parasitism. RT-qPCR analysis showed that the relative expression patterns of the six target olfactory genes varied considerably among the parasitic stages of the D. helophoroides larvae (i.e., neonates and days 2–6 of parasitism; Fig. 1). Three of the six genes were significantly upregulated at the neonatal stage compared with the parasitic stages: DhelCSP2 (F¼ 549.400; df ¼ 5, 12; P < 0.001), DhelOR2 (F ¼ 21.536; df ¼ 5, 12; P < 0.001), and DhelIR2 (F ¼ 136.728; df ¼ 5, 12; P < 0.001; Fig. 1). The remaining three genes were significantly upregulated on days 4 and 5 postparasitism compared with day 0: DhelOBP8 (F ¼ 20.356; df ¼ 5, 12; P < 0.001), DhelGR5 (F ¼ 114.373; df ¼ 5, 12; P < 0.001), and DhelSNMP1 (F ¼ 228.312; df ¼ 5, 12; P < 0.001; Fig. 1).



Citation: Journal of Entomological Science 58, 3; 10.18474/JES22-64
Relative expression patterns of olfactory genes in the heads and thoracoabdominal parts of D. helophoroides larvae. We also identified significant differences in the relative expression levels of five of the six target olfactory genes between the heads and thoracoabdominal parts of D. helophoroides neonates by using RT-qPCR. Four genes were significantly upregulated in the head compared with the thoracoabdominal area: DhelOBP8 (t ¼ 15.115; df ¼ 2; P ¼ 0.004), CSP2 (t ¼ 15.068; df ¼ 2; P ¼ 0.004), DhelOR2 (t ¼ 5.404; df ¼ 2; P ¼ 0.033), and DhelIR2 (t ¼ 11.937; df ¼ 2; P ¼ 0.007; Fig. 2). Conversely, DhelGR5 was significantly downregulated in the head compared with the thoracoabdominal area (t ¼ –10.099; df ¼ 2; P ¼ 0.010; Fig. 2). The sixth gene, DhelSNMP1, was upregulated in the head compared with the thoracoabdominal area, but this upregulation was not significant (t ¼ 3.580; df ¼ 2; P ¼ 0.070; Fig. 2).



Citation: Journal of Entomological Science 58, 3; 10.18474/JES22-64
RNAi knockdown efficiency. Based on transcription levels before and after dsRNA treatment, RNAi efficiently knocked down four of the six target olfactory genes in D. helophoroides neonates. The genes significantly downregulated after dsRNA treatment were DhelOBP8 (t ¼ 42.220; df ¼ 2; P ¼ 0.001), DhelCSP4 (t ¼ 11.912; df ¼ 2; P¼ 0.007), DhelOR2 (t¼ 10.856; df ¼ 2; P¼ 0.008), and DhelSNMP1 (t ¼ 19.815; df ¼ 2; P ¼ 0.003; Fig. 3). The remaining two target genes were also downregulated, but not significantly: DhelIR2 (t ¼ 4.115; df ¼ 2; P ¼ 0.054), and DhelGR5 (t ¼ 2.903; df ¼ 2; P ¼ 0.101; Fig. 3).



Citation: Journal of Entomological Science 58, 3; 10.18474/JES22-64
Discussion
Insects rely on a keen sense of olfaction to perceive various chemical signals in the natural environment (Sachse and Krieger 2011). Dastarcus helophoroides mainly use host volatiles and host-associated plant volatiles to locate the host M. alternatus (Lu et al. 2012, Wei et al. 2009, Yu 2014). Examination of the parasitism-related expression patterns of the six representative olfactory genes included in this study showed that DhelCSP2, DhelOR2, and DhelIR2 were significantly upregulated before the initiation of parasitism, suggesting that these three genes might play crucial roles in host recognition by D. helophoroides larvae. Conversely, DhelOBP8, DhelGR5, and DhelSNMP1 were significantly and strongly upregulated 4–5 d after parasitism initiation, suggesting that these genes might be involved in parasitic functions in D. helophoroides larvae other than or as well as locating the host.
Four of the six representative olfactory genes included in this study were significantly upregulated in the heads of D. helophoroides neonates compared with the thoracoabdominal area, suggesting that the primary functions of DhelCSP2, DhelOR2, DhelOBP8, and DhelIR2 were concentrated in the head. However, DhelGR5 was significantly upregulated in the thoracoabdominal area compared with the head, suggesting that the primary functions of this gene may be nonolfactory. Indeed, all six olfactory genes were constitutively expressed in both the heads and the thoracoabdominal area of D. helophoroides larvae, suggesting that all six genes may play both olfactory and nonolfactory roles. Similar results have been reported in other insects. For example, OBPs and CSPs may be involved in development as well as the olfactory system in Apis mellifera L. (Hymenoptera: Apidae) (Forêt et al. 2007, Maleszka et al. 2007). In addition, the OBP of Phormia regina (Meigen) (Diptera: Calliphoridae) binds long-chain fatty acids and contributes to nutrient uptake (Ishida et al. 2013). However, further investigations of the nonolfactory functions of insect olfactory genes are required.
RNAi is a posttranscriptional gene-silencing mechanism in eukaryotic cells that is a potent tool for studying gene function (Wang et al. 2018). Herein, gene-specific dsRNAs were successfully delivered to D. helophoroides neonates by immersion, resulting in the significant knockdown of four of the six target olfactory genes. Our results demonstrate the effectiveness of RNAi in D. helophoroides larvae, providing a framework for further functional explorations of D. helophoroides olfactory genes, which may lead to the development of improved biological pest control methods using D. helophoroides.

Relative expression levels of olfactory genes in Dastarcus helophoroides larvae at different parasitic stages: neonates (N) and days 2–6 after the initiation of parasitization. Actin was used as the internal reference. Values graphed represent means ± SE of three independent replicates. Lowercase letters above the bars indicate significant differences in expression levels among parasitic stages (P ≤ 0.05, analysis of variance).

Relative expression levels of olfactory genes in the heads and thoracoabdominal parts of Dastarcus helophoroides neonates. Actin was used as the internal reference. Values graphed represent means ± SE of three independent replicates. Asterisks above the bars indicate a significant difference in expression level between the head and the thoracoabdominal part (P < 0.05, pairwise t test).

Knockdown efficiency of double-stranded RNA (dsRNA) treatments in Dastarcus helophoroides neonates. Actin was used as the internal reference. Values graphed represent means ± SE of three independent replicates. Asterisks above the bars indicate a significant difference in expression level between the target olfactory gene (DhelOBP8, DhelCSP2, DhelOR2, DhelIR2, DhelGR5, or DhelSNMP1) and the control gene (enhanced green fluorescent protein [EGFP]) after dsRNA treatment (P < 0.05, pairwise t test).
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