Introduction

Insects use their sense of smell to find mates for reproduction and to locate hosts for nutrition by detecting semiochemicals. These compounds enter the antennae by diffusion through pores on the cuticle and are transported to the olfactory neurons across the sensillum lymph (Zhou et al. 2010). Several functional components of the insect olfaction system, which are involved in olfactory signal recognition and transduction in insects, are currently under investigation at the molecular level. These include odorant binding proteins (OBPs), sensory neuron membrane proteins (SNMPs), and olfactory receptors (ORs) (Benton et al. 2007; Jin et al. 2008). Insect OBPs are thought to provide the first filtering mechanism for semiochemicals and to mediate the activation of ORs (Grosse-Wilde et al. 2006; Pophof 2004; Syed et al. 2006). Recent studies at Rothamsted (Hooper et al. 2009; Zhou et al. 2009) now provide a starting point for the potential use of OBPs as targets to interfere with insect host location and mating behavior. Such non-insecticidal approaches could play a role as part of integrated pest management strategies and broaden the arsenal of available tools for insect pest control. OBPs include pheromone binding proteins and general odorant binding proteins, both of which have molecular masses of approximately 15 kDa, a low isoelectric point, and six conserved cysteine residues paired in three disulfide bridges (Leal et al. 1999; Scaloni et al. 1999). Pheromone binding proteins (PBPs) are expressed mainly in male insect antennae, and are thought to be involved in sex pheromone detection. General odorant binding proteins (GOBPs) are differentiated into GOBP1 and GOBP2 based on amino acid sequence homology, and are capable of binding more general odorants, such as green leaf volatiles (Vogt et al. 1991).

Studying the odorant-binding characteristics of insect OBPs will help understand their physiological functions (Sun et al. 2012). The odorant-binding characteristics of OBPs can be determined using many techniques, such as isotope labeling methods, volatile odorant-binding assays, and fluorescence binding assays (Briand et al. 2001; Gu et al. 2012; Vogt and Riddiford 1981). Among these methods, a fluorescence binding assay is most widely used, with 1-aminoanthracene, 1-anilinonaphthalene-8-sulfonic (ANS) and N-phenyl-1-naphthylamine (1-NPN) often selected as fluorescent probes. The OBPs used in a fluorescence binding assay usually are obtained by prokaryotic expression. Several studies have investigated the odorant-binding characteristics of OBPs by using the fluorescence binding assay. Liu et al. (2012) conducted tests against Orthaga achatina (Butler) (Lepidoptera: Pyralidae) to determine the affinity of GOBP2, and revealed that GOBP2 could not bind some plant volatiles, such as farnesol and farnesene, but instead displayed higher binding affinities for the putative sex pheromones than OachPBP1. The results suggested that OachGOBP1 has possible functions in sex pheromone reception. In the parasitic wasp, Microplitis mediator Haliday (Hymenoptera: Braconidae), OBP1 to OBP7 can bind various odorant molecules with different binding affinities, in which they show high affinity for the flowering plant volatile, β-ionone (Zhang et al. 2009). MmedOBP5 failed to bind any of the other organic compounds tested, except β-ionone (Zhang et al. 2011). The binding properties of 114 odorants to Adelphocoris lineolatus OBP1 were measured by using 1-NPN as a fluorescent probe. The results revealed that AlinOBP1 has high binding affinities with two major putative pheromone components, namely, ethyl butyrate and trans-2-hexenyl butyrate. In addition, AlinOBP1 can bind six volatiles from cotton, namely octanal, nonanal, decanal, 2-ethyl-1-hexanol, β-caryophyllene, and β-ionone (Gu et al. 2011). GOBP2 protein of Chilo suppressalis has significant affinity to cis-11-hexadecenal (Z11-16: Ald), the main component of C. suppressalis pheromone and to laurinaldehyd and benzaldehyde, two general plant volatile aldehydes (Gong et al. 2009).

Microplitis mediator is a polyphagous solitary larval endoparasitoid that is widely distributed from Central Europe to China and attacks approximately 40 species of Lepidoptera, including Helicoverpa armigera, Mythimna separata, and Barathra brassicae. (Arthur and Mason 1986; Shenefelt 1973). In our previous study, the odorant-binding characteristics of seven recombinant OBPs (MmdeOBP1-7) in M. mediator were investigated by using fluorescence binding experiments with bis-ANS as a fluorescent probe. All seven M. mediator OBPs can bind various odorant molecules, among which, β-ionone has the best affinity (Zhang et al. 2011). Zhang et al. (2009) also investigated the expression pattern of the MmedOBP1-7 genes by real-time polymerase chain reaction (PCR). In this study, we identified three new putative OBP genes, MmedOBP8-10 (GenBank No. JF738026 for MmedOBP8, JN587220 for MmedOBP10; the GenBank No. for MmedOBP9 has not been released yet) from a previous M. mediator antennal cDNA library. Quantitative real-time PCR (qRT-PCR) analysis revealed that all three of the OBP genes were expressed mainly in the antennae of adult wasps. We also investigated their odorant-binding characteristics by using fluorescence binding assay with 1-NPN as the fluorescent probe. Y-tube olfactometer assays were conducted to verify whether the odorant molecules that exhibit high binding affinities with MmedOBP8-10 in the fluorescence binding assay can elicit a behavioral response in adult M. mediator.

Methods and Materials

Insects and Tissue Collection

A colony of M. mediator was obtained from the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry, China. The wasps emerged from the host as mature larvae and spun a silk cocoon outside the larvae on which they pupated. Emerged adults of M. mediator were fed a 30 % honey solution in a growth chamber programmed at 28 ± 1 °C, 75 % relative humidity, and 16:8 (L:D) photoperiod.

The female antennae, male antennae, head (without antennae), thorax, abdomen, legs, and wings were excised from 2-3-d-old adult wasps, immediately frozen in liquid nitrogen and stored at −80 °C until use.

RNA Extraction and cDNA Synthesis

Total RNA from each sample was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The integrity of the RNA samples was assessed by 1.1 % agarose electrophoresis, and the concentration was estimated by spectrophotometry at 260 nm. Before transcription, total RNA was treated with RQ1 RNase-Free DNase at 37 °C for 30 min (Promega, Madison, WI, USA) to remove residual genomic DNA. Single-stranded cDNAs were synthesized using the SuperScriptTM III Reverse Transcriptase System (Invitrogen). All procedures were conducted following the manufacturer’s instructions.

Quantitative Real-Time PCR (qRT-PCR)

Tissue distributions of the three OBP transcripts were measured by qRT-PCR by using the ABI Prism 7500 Fast Detection System (Applied Biosystems, Carlsbad, CA, USA). The β-actin gene (GenBank accession number: KC193266) of M. mediator was used as the endogenous control to normalize target gene expression and correct for sample-to-sample variation. The primers (Table 1) of the target and the reference genes were designed using Primer Express 3.0 (Applied Biosystems). The RNA/cDNA preparation of each tissue was repeated three times. Quantitative Real-Time PCRs were conducted in 20 μl reaction volumes, each containing 10 μl of 2× SYBR Premix Ex Taq (TaKaRa, Dalian, China), 0.5 μl of each primer (10 μM), 0.4 μl of Rox reference II (50×), 1 μl of sample cDNA (200 ng/μl), and 7.6 μl of sterilized H2O. The qRT-PCR cycling conditions were as follows: 95 °C for 30 s; 40 cycles of 95 °C for 5 s, and 60 °C for 30 s; melt curves stages at 95 °C for 15 s; 60 °C for 1 min; and 95 °C for 15 s. The experiments for the test samples, endogenous controsl and negative controls were performed in triplicate to ensure reproducibility. Relative quantification was performed using the comparative 2-ΔΔCt method (Livak and Schmittgen 2001). All data were normalized to endogenous β-actin levels from the same tissue samples.

Table 1 Oligonucleotide primers used in the study
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Recombinant Protein Expression and Purification

Full-length sequences of the MmedOBP8-10 genes were obtained by screening the M. mediator antennal cDNA library and were analyzed using the Gene Explore 1.0 software. The open reading frames (ORFs) of MmedOBP8-10 without signal peptides were obtained by the Signal P 3.0 Server. The molecular weight and isoelectric point of the MmedOBP8-10 proteins were predicted in http://www.expasy.org/tools/pi-tool.html. Table 1 lists the specific primers for PCR amplification and protein expression. The sample cDNA of the antennae was used as the template for the PCR amplification. The PCR product was first cloned into a pGEM-T Easy vector (Promega, Madison, WI, USA) and then excised and cloned into an expression vector PET-30a (+) for overexpression in prokaryotic BL21 (DE3) cells (Takara, Dalian, China). Protein expression was induced by adding isopropylthio-β-galactoside (IPTG) at a final concentration of 0.2 mM when the culture reached an optical density of 0.8. Cells were grown for 6 h at 30 °C, harvested by centrifugation, and sonicated. The recombinant proteins present in the supernatant of the lysate were purified by Ni ion affinity chromatography. The His-tag was removed by using recombinant enterokinase (Xinbainuo, Shanghai, China). By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the sizes of the MmedOBP8-10 proteins were checked. The concentration of the purified protein was measured by the Bradford method using bovine serum albumin as the standard.

Fluorescence Binding Assay

The fluorescence measurements were performed according to the method of Gu et al. (2011) on a fluorescence spectrometer (F-380, Tianjin, China) in a 1 cm light path quartz cuvette. The slit width used for the excitation and emission was 10 nm. 1-NPN was dissolved in methanol from a 1 mM stock solution. The fluorescent probe 1-NPN was excited at 365 nm, and the emission spectra were recorded from 350 nm to 500 nm. Forty five small organic compounds were used in the fluorescence binding assay (Table 2). A 2 μM solution of the protein in 50 mM Tris–HCl (pH 7.4) was titrated with aliquots of 1 mM 1-NPN to a final concentration of 2 μM to 20 μM to measure the affinity of 1-NPN to MmedOBP8-10. Each experiment was repeated three times. The affinities of each OBP for the 45 small organic compounds were measured by a competitive binding assay using both 1-NPN and MmedOBP8-10 at 2 μM by adding ligands (2 μM to 16 μM). The binding data were collected as three independent measurements.

Table 2 Company, CAS number and purity of all of the chemicals tested
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Behavioral Trials

Behavioral responses of M. mediator to the small organic compounds that exhibited high binding affinities with MmedOBP8-10 in the fluorescence binding assay were tested at room temperature (22 °C to 25 °C) in a Y-tube olfactometer equipped with two-armed glass tubes (2 cm i.d.) with an air flow of 300 ml/min. The system consisted of a 15 cm-long central tube and two 15 cm-long lateral arms. The angle between the lateral arms was 120°. A newly emerged male or female M. mediator adult was placed at the end of the central tube. A filter paper strip was immersed in the solutions of the odor molecules to be tested at the dose of 10 μl/ml, and placed in one of the arms of the Y-tube olfactometer. The other arm contained a filter paper strip that had been immersed in paraffin oil, serving as the control. The initial response of the M. mediator adult, identified as walking/flying into one of the arms and remaining there for at least 15 s, was recorded. If the M. mediator adult did not make a choice within a minute of its release, it was removed and the response was recorded as ‘no choice’. After five M. mediator individuals were tested, the two arms were exchanged to avoid the asymmetric bias effect. To minimize the visual distraction for M. mediator, the Y-tube olfactometer was placed inside a white paper box that was open on the top for illumination and on the front side for observation. The box was illuminated by vertically hanging an office lamp (20 W, 250 Lux) 50 cm above the olfactometer tube. After ten wasps were observed, the old impregnated filter paper strip was discarded and replaced with a new impregnated filter paper strip. Next, the olfactometer set-up was washed with soapy water, rinsed with acetone, and air-dried. Sixty adults of M. mediator (including 30 males and 30 females) in each treatment group were observed, and each wasp was used only once.

Data Analysis

To measure the affinity of the fluorescent ligand 1-NPN to each MmedOBP, the intensity values corresponding to the maximum of fluorescence emission were plotted against the total ligand concentrations. The ligand-binding properties were evaluated by using the fluorescence intensities, assuming that the protein was 100 % active, with a stoichiometry of 1: 1 (protein: ligand) at saturation. The curves were made linear using Scatchard Plots. The dissociation constants (KD) of the competitors were calculated from the corresponding IC50 values using the equation: KD = [IC50]/(1 + [1-NPN]/K1-NPN), where [1-NPN] is the free concentration of 1-NPN and K1-NPN is the dissociation constant of the complex protein/1-NPN.

Data from the qRT-PCR tests were analyzed using SAS 9.0 (SAS 9.0 system for windows, 2002, SAS Institute Inc., Cary, NC, USA). The behavioral assay data also were analyzed by a chi-squared test using the SAS 9.0 software. ANOVA and Duncan’s new multiple range test (P = 0.05) were used to determine whether differences in the OBP mRNA levels were significant among different treatment groups.

Results

Tissue Expression of MmedOBP8-10 Transcripts

We examined the tissue-specific expression level of the MmedOBP8-10 genes in various tissues of adult M. mediator wasps by qRT-PCR (Fig. 1). All three OBP genes were expressed mainly in the antennae. MmedOBP8 and MmedOBP9 have significantly higher levels of transcript in the female relative to male antennae, whereas MmedOBP10 showed higher levels of expression in the male compared with female antennae. In addition, MmedOBP10 displayed very low levels of expression in the legs, wings, and thorax. The expression profiles of MmedOBP8-10 indicated that the three OBPs may play roles in chemoreception in M. mediator.

Fig. 1
figure 1

Expression profile of MmedOBP8-10 using qRT-PCR. The standard errors of the means of three biological replicates are represented by error bars. Different letters within the same figure indicate that the expression levels were significantly different (P < 0.05)

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Expression and Purification of MmedOBP8-10

The lengths of the predicted ORFs of MmedOBP8-10 were 459, 420, and 393 bp, which encoded 153, 140, and 131 amino acids, respectively. The predicted protein molecular weight and isoelectric point of MmedOBP8-10 were 14.72 kDa and 4.9, 15.94 kDa and 5.08, and 19.16 kDa and 5.36, respectively. The SDS-PAGE analysis showed that the LB fluid nutrient medium that contained the PET-30(a)/MmedOBP8-10 BL21 bacteria produced the target protein bands with IPTG induction. The three target proteins were expressed primarily in the supernatant (Fig. S1). The MmedOBP8-10 proteins were purified using Ni ion affinity chromatography and were tested for their binding properties after removing the His-tag using enterokinase.

Fluorescence Binding Assay

The three purified proteins were used in a fluorescence competitive binding assay with 1-NPN as the fluorescent probe and 45 small organic compounds as the competitors. The small organic compounds that were selected comprised eight alkanes, ten alcohols, seven aldehydes, six ketones, five esters, two aromatic compounds, and seven terpenes. First, we calculated the KD values of the MmedOBP/1-NPN complexes according to the changes in the fluorescence intensity. The values used to calculate the KD of competitors were 3.07, 2.48 and 5.46 μm for MmedOBP8, MmedOBP9, and MmedOBP10, respectively (Fig. 2).

Fig. 2
figure 2

Binding curve of 1-NPN to MmedOBP8 and Scatchard plot (insert). A 2 mM solution of the protein in Tris buffer was titrated with a 1 mM solution of 1-NPN in methanol to final concentrations of 2 μM to 20 μM. Binding curves were also determined for MmedOBP9 and MmedOBP10 (data not shown), and the binding constants of the MmedOBP/1-NPN complex were 3.07, 2.48, and 5.46 μM for MmedOBP8, MmedOBP9, and MmedOBP10, respectively

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The binding affinities of the three MmedOBPs to the small organic compounds are listed in Table 3. Distinct binding affinities were observed for the three proteins. MmedOBP8 bound to ten ligands, MmedOBP9 to eighteen ligands, and MmedOBP10 to nine ligands. MmedOBP8-10 all bound strongly to one of the eight alkanes, namely nonane, with KD values of 4.89, 3.83, and 7.65 μM, respectively. The three MmedOBPs also showed strong binding affinities to two of the ten alcohols, namely, farnesol and nerolidol, with KD values ranging from 1.32 μM to 29.73 μM. MmedOBP8-10 showed medium binding affinities to seven aldehydes and six ketones. In contrast, β-ionone bound with high affinity to all three MmedOBPs, with KD values of 3.43, 2.47, and 7.14 μM, respectively. MmedOBP8-10 showed high binding affinities to acetic ether among the five esters, with KD values of 6.21, 4.46, and 10.38 μM, respectively. MmedOBP8-10 also showed high binding affinities to one of the seven terpenes, namely, farnesene, with KD values of 8.39, 1.80 and 12.47 μM, respectively. Two aromatic compounds (benzaldehyde and methyl phenylacetate) failed to bind any of the three MmedOBPs.

Table 3 Binding affinities of 45 chemical compounds to MmedOBP8-10 proteins
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Behavioral Trials

The compounds that exhibited high binding affinities with the MmedOBPs were selected for the behavioral trials. Five of the selected compounds could elicit behavioral responses in M. mediator adults (Fig. 3). The parasitoids showed a significant attraction to β-ionone, nonanal and farnesene (χ 2 β-ionone = 5.14, P = 0.027; χ 2 nonanal = 6.87, P = 0.018; χ 2 farnesene = 4.76, P = 0.020). However, the parasitoids showed a significant repulsion behavior to nonane and farnesol (χ 2 nonane = 7.46, P = 0.026; χ 2 farnesol = 5.89, P = 0.038). Nerolidol and acetic ether were neither attractive nor repulsive to the parasitoids (χ 2 nerolidol = 1.89, P > 0.05; χ 2 acetic ether = 1.26, P > 0.05).

Fig. 3
figure 3

Behavioral response of Microplitis mediator in a Y-tube olfactometer bioassay when given a choice between different odorant chemicals (odor stimulus) and paraffin oil (control). χ 2 tests were used to determine significant differences in the number of parasitoids choosing a particular odor, with levels of significance: *P < 0.05. ns, not significant

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Discussion

In our previous work, we found that MmedOBP2, MmedOBP4, MmedOBP5, and MmedOBP7 were expressed only in the antennae, whereas MmedOBP3 and MmedOBP6 were ubiquitously expressed in all of the tissues examined except the antennae (Zhang et al. 2009). These findings could help to understand the olfactory processes in parasitoids when finding their host. In the present research, the tissue distribution results indicated that MmedOBP8-10 were mainly expressed in the antennae. We suspected that MmedOBP8-10 may play crucial roles in the chemoreception of M. mediator. Therefore, we focused on the binding characteristics of these OBPs and their relationship with volatiles.

Among the 45 compounds tested in binding assays, eight were from cotton (Yu et al. 2006, 2007, 2010), and five were from maize (De Moraes et al. 1998; Yan et al. 2005). Most compounds used in our experiments were typical volatiles from the host plants. The results of the fluorescence competitive binding assays showed that MmedOBP8-10 had a broad odorant-binding ability being capable of binding alkanes, alcohols, aldehydes, ketones, esters, and terpenes. The three OBPs (MmedOBP8-10) showed high binding affinities for β-ionone. This result is consistent with previous research by Zhang et al. (2011), who reported that all seven OBPs (MmedOBP1-7) have high binding affinities with β-ionone. Nonanal and farnesol did not bind to any of MmedOBP1-7 (Zhang et al. 2011). However, in this study, both nonanal and farnesol exhibited high binding affinities with MmedOBP8-10. The compound, 2-hexanone, a green leaf volatile, aids herbivorous insects in locating hosts and looking for nutritional supplements (Du et al. 2001). MmedOBP8-10 failed to bind to 2-hexanone in the present experiment. However, in a previous study, MmedOBP4 showed a modest binding affinity to 2-hexanone, with a KD value of 25.14 μM (Zhang et al. 2011). These differences are probably caused by the functional differentiation among the M. mediator OBPs. MmedOBP9 also showed a high binding affinity to the sex pheromone analog ethyl butyrate, with a KD value of 7.91 μM, which indicated that the OBPs of M. mediator can simultaneously perceive sex pheromones and host volatiles.

The MmedOBP8-10 proteins exhibited high binding affinities to β-ionone, nonanal, farnesene, nonane, and farnesol in the fluorescence competitive binding experiments. These five compounds elicited a significant behavioral response from M. mediator in the behavioral trials. Given that M. mediator was significantly attracted to β-ionone, nonanal, and farnesene, these three compounds could be selected for developing slow-release agents to attract M. mediator and improve the searching efficiency for strategies to eliminate insect pests. Because several functional components including OBPs, SNMPs, and ORs are involved in olfactory signal recognition and transduction in insects, the results of the fluorescence competitive binding assay did not always correlate well with the biological activity of the ligands tested. In this work, the MmedOBP8-10 proteins showed high binding affinities to nerolidol and acetic ether, but M. mediator did not respond to the two compounds. For future studies, we will conduct RNAi experiments or similar assays to check the roles of MmedOBPs in the detection of the compounds and influence on the behavior of M. mediator.

To sum up, we successfully expressed three MmedOBPs in Escherichia coli and purified them by Ni ion affinity chromatography. We characterized the ligand-binding specificities of these OBPs to 45 diverse small organic compounds. In addition, we verified the biological activity of the odorant molecules that exhibited high binding affinities with MmedOBP8-10 in the fluorescence competitive binding experiments for M. mediator by Y-tube assays. The present results will provide insight into the mechanism of olfactory recognition when parasitoids search for and locate their hosts.