Olfaction plays a major role in insects where it assists the insect in locating host and food plants, find mates, identify oviposition sites, and avoid predators (Hallem et al. 2006, Wu et al. 2017). The antennae of insects possess thousands of sensillae with different morphological and functional types, including trichodea, chaetica, basiconica, coeloconica, styloconica, auricillica, and squamiformia. Sensilla trichodea are the most abundant olfactory sensillae on the antennae (Gómez et al. 2003, Zacharuk and Shields 1991). Odorant molecules enter olfactory sensillae through pore tubules in the plasma membranes, passing into the lymph around the olfactory receptor neurons (Carey and Carlson 2011). The olfactory process of insects has been suggested to contain several major peripheral olfactory proteins, including odorant receptors (ORs), odorant-binding proteins (OBPs), chemosensory proteins (CSPs), pheromone-binding proteins (PBPs), ionotropic receptors, sensory neuron membrane proteins, and odorant-degrading enzymes (Bian et al. 2018, Jacquin-Joly and Merlin 2004, Leal 2013, Wang et al. 2021). Insects mainly depend on ORs for the detection of odorant molecules (An et al. 2016).

Compared with mammalian ORs, insect ORs have evolved an opposite membrane topology from that of G-protein coupled receptors where the N- and C-terminus are intracellular and extracellular, respectively (Benton et al. 2006, Hansson and Stensmyr 2011). A functional OR is a heterodimeric complex composed of a poorly conserved ligand-binding receptor (OR) and a conserved co-receptor (Orco, formerly Or83b) (Benton 2009). The ligand-binding ORs are classified into pheromone receptors, gustatory receptors, and general ORs (Fleischer et al. 2018). General ORs are used for the detection of plant volatile compounds (Tanaka et al. 2009).

Grapholita molesta Busck (Lepidoptera: Tortricidae), commonly known as the oriental fruit moth, causes severe damage worldwide to pome and stone fruits (Barros-Parada et al. 2018, Wei et al. 2015). The first- and second-generation larvae damage extremely young shoots of peach (Prunus persica [L.] Batsch), nectarine (P. persica var. nectarina), and plum (Prunus salicina Lindl) trees, while the third- and fourth-generation larvae mainly feed on the pulp, including that of apple (Malus pumila Borkhausen) and pear (Pyrus sp,) (Duarte et al. 2015). The adults can switch to a different host by detecting and following changes in host-plant volatiles (Hughes and Dorn 2002, Siegwart et al. 2015). To control G. molesta, it may be useful to target host-plant volatile reception by targeting OR genes. The G. molesta antennal transcriptome is available, and many olfaction genes have been identified, including 48 ORs, 28 OBPs, and 17 CSPs (Li et al. 2015). Several OBPs, PBPs, and CSPs have been studied (Chen et al. 2018; Li et al. 2016, 2019; Song et al. 2014; Zhang et al. 2012). The ORs of G. molesta, however, are poorly understood. In this study, GmolOR7 was identified and evaluated to determine its expression patterns in G. molesta. The results will provide an indication for further functional study of the GmolOR7 in G. molesta.

Materials and Methods

Insects. Grapholita molesta were supplied by the College of Plant Protection, Northwest A&F University (Yangling, Shaanxi, China). Larvae and adults were fed on an artificial diet and with a 5% honey solution, respectively. All insects were reared at 25 ± 1°C, 70 ± 10% relative humidity, and a 15:9 h (L:D) photoperiod (Du et al. 2010).

Samples, RNA extraction, and cDNA synthesis. Grapholita molesta samples were obtained from various stages of development (eggs; 1st- to 5th-instar larvae; pupae; and 1-d-old adults, both male and female), antennae of 1-, 3-, 5-, and 7-d-old adult females, and different tissues of adults, including antennae, heads without antennae, thoraxes, abdomens, wings, and legs. Tissues were frozen in liquid nitrogen and subsequently stored at –80°C. RNAiso Plus was used for extraction of total RNA (TaKaRa, Dalian, China), and concentration and quality were evaluated by a SimpliNano spectrophotometer (GE Healthcare, London, U.K.) and 1% agarose gel electrophoresis. The RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA) was used for first-strand cDNA synthesis from 1 µg of total RNA following the kit instructions and stored at –20°C.

Molecular cloning. Referring to antennal transcriptome of adult female of G. molesta (Li et al. 2015), the GmolOR7 ORF (open reading frame) was cloned using 3′ RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) and ordinary PCR. For 3′ RACE, the 3′ region of GmolOR7 was amplified as previously described. Synthesis of primers was done by Sangon Biotech (Shanghai, China). The RACE primers were 3′Outer-F: 5′-TTAGTATGCGTGATGATGGA-3′, 3′Outer-R: 5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3′; 3′Inner-F: 5′-TGCGTGATGATGGAGTTG-3′, 3′Inner-R: 5′-CTAATACGACTCAC-TATAGGGC-3′. The 20-µL reaction contained 10 µL Premix Taq™ (TaKaRa), 1 µL each primer (10 µM), 1 µL cDNA, and 7 µL RNase-free water. Conditions for the first amplification step were: four cycles at 98°C for 30 s, 64°C for 1 min, 72°C for 2 min; after which the annealing temperature was reduced by 2°C for four cycles. A further 25 cycles at 98°C for 30 s, 56°C for 1 min, 72°C for 2 min, and 72°C for 10 min were performed. The second step was the same as the first step. A Universal DNA Purification Kit (TianGen, Beijing, China) was used to purify the PCR products which were then ligated into the pMD™-19T cloning vector (TaKaRa) and transformed into DH5α competent cells (TianGen). Sequencing of positive clones was done by Sangon Biotech. The primers OR7-F: 5′-ACAGATACTACCAT-GAAAAA-3′, OR7-R: 5′-TTATCCTTTGTTCAGTGTC-3′ containing the start and stop codons were used for GmolOR7 ORF amplification.

Quantitative real-time PCR (qPCR). GmolOR7 expression in the different tissues and stages was evaluated by qPCR with β-actin as reference (GenBank number: KF022227.1). The specific primers were as follows: qOR7-F: 5′-GCAGCCTACCCAGTATGGTC-3′, qOR7-R: 5′-TGGGAAGTGAGATCCGAAAC-3′; qActin-F: 5′-TGCGTGACATCAAGGAGAAG-3′, qActin-R: 5′-TACCGATGGT-GATGACCTGA-3′. The reaction system contained 10 µL of SYBR^® Premix Ex Taq™ II (TaKaRa), 0.8 µL of each primer (10 µM), 2 µL of cDNA, and 6.4 µL of RNase-free water in 20 µL total volume. Triplicate amplifications were conducted on the Bio-Rad CFX96 Real-Time System (Bio-Rad, Hercules, California, USA) as following: 95°C for 30 s, 40 cycles of 95°C for 10 s, 59°C for 30 s, and 72°C for 30 s. Measurements every 0.5°C during the 59–95°C cycle were used to generate melting curves to verify primers. The standard curve was used to evaluate the efficiency of amplification using a 5-fold dilution series of cDNA. Three biological replicates from each sample were used to calculate the relative sample expressions of GmolOR7 were determined using the 2^–ΔΔCt method (Livak and Schmittgen 2001).

Sequence and data analysis. GmolOR7 orthologs were identified by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The TMHMM Server v 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) was used for transmembrane domain prediction, and the Compute pI/Mw tool (https://web.expasy.org/compute_pi/) was used for the prediction of molecular weight and isoelectric point. Primers were designed by Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/#PRIMER_SEQUENCE_INPUT). Sequence alignment and comparisons were done using DNAMAN 6.0 and ClustalX 2.1 software. Phylogeny was evaluated by MEGA 6 software using the neighbor joining method with bootstrap values based on 1,000 replicates at the nodes. Data were analyzed using SPSS 22, and figures were plotted using Origin 2021.

Results

Identification of G. molesta GmolOR7. The GmolOR7 (GenBank accession number: MH844555) cDNA contained an ORF of 1,125 base pairs, which encoded a protein of 374 amino acids. The molecular weight of the protein was 41.86 kDa and its theoretical isoelectric point of 6.70. Eight transmembrane domains were predicted by sequence analysis with the TMHMM Server v 2.0; the N-terminus was predicted to be intracellular and the C-terminus extracellular (Fig. 1).

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Sequence analysis of G. molesta GmolOR7. Sequence alignment showed that the ORF regions in GmolOR7 and Cydia pomonella OR54 (CpomOR54) shared 80.21% sequence identity (Fig. 1). As expected, GmolOR7 and CpomOR54 were in the same phylogenetic clade and were closely related (Fig. 2).

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Expression profiles of G. molesta GmolOR7. GmolOR7 was expressed in all developmental stages from eggs to adults, with especially high expression in adults where females showed markedly higher levels than males (P < 0.05) (Fig. 3). Expression was predominant in the antennae in both sexes, in comparison with other tissues (heads, thoraxes, abdomens, legs, and wings). Moreover, GmolOR7 expression in female antennae was significantly higher than that in males (P < 0.01). GmolOR7 also expressed in other tissues, although the levels were very low (Fig. 4). Adult females of all ages showed high levels of expression in antennae, with the highest expression seen in 3-d-old female adults (P < 0.05) (Fig. 5).

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Discussion

ORs are responsible for detecting host-plant volatiles, indicative of suitable oviposition sites and sources of food (Jordan et al. 2009). Here, a general OR gene GmolOR7 was cloned from the oriental fruit moth. GmolOR7 was found to have eight transmembrane domains (Fig. 1), which is consistent with the typical characteristics of insect ORs (An et al. 2016, Bengtsson et al. 2012). GmolOR7 was presented a low homology across several insect species, which was consistent with the previous studies (Hallem et al. 2006, Robertson et al. 2003). Previously, the different ORs were clustered into diverse branches (Chen et al. 2020b, Zhang et al. 2013). In our phylogenetic tree, GmolOR7 was classified into Lepidoptera (Fig. 2), confirming that providing a foundation for study of the correspondence between different insect species and OR functions.

GmolOR7 was found to be highly expressed in adults (Fig. 3), particularly in the antennae with transcript levels higher in female antennae than male (Fig. 4). The expression profiles of GmolOR7 were very similar to those of other insect ORs (Sun et al. 2019, Yan et al. 2015, Zhang et al. 2019), suggesting the role of this gene in detecting plant volatiles. Three-day-old females showed the highest expression of GmolOR7 in their antennae (Fig. 5). Previous studies showed that peak flight, mating, and oviposition activities are maximal in individuals 3 d after eclosion (Hughes and Dorn 2002, Zhang and Wu 2012). This indicates the vital role of GmolOR7 in controlling female moth behaviors, such as host-plant location and oviposition site selection.

Generally, diverse OR genes display different functions in the same insect species. For example, BmorOR56 of Bombyx mori L., AlucOR30 of Apolygus lucorum (Meyer-Dur), and AlepOR3 of Athetis lepigone (Möschler) specifically respond to cis-jasmone, (1S)-(-)-verbenone, and Z7-12:AC, respectively. Nevertheless, BmorOR24 of B. mori and AlucOR28 of Apolygus lucorum broadly tune to several volatiles, and AlepOR4 of Athetis lepigone responds to five sex pheromones (Tanaka et al. 2009, Yan et al. 2015, Zhang et al. 2019). In G. molesta, GmolOR9 and GmolOR12 responded to eight and five host-plant volatiles, respectively. GmolOR20 and GmolOR21 had no responses to any of the odorant compounds tested (Chen et al. 2020a, 2020b). Consequently, GmolOR7 may be involved in the recognition of host-plant volatiles, and GmolORs and GmolOR7 have different functions.

In summary, GmolOR7 shows similar functional properties to general ORs. These results improve the understanding of OR characteristics and provide an indication for further functional study of the GmolOR7 in G. molesta.