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The antennal transcriptome analysis and characterizations of odorant-binding proteins in Megachile saussurei (Hymenoptera, Megachilidae)

BMC Genomics volume 24, Article number: 781 (2023) Cite this article

Abstract

Background

Odorant-binding proteins (OBPs) are essential in insect’s daily behaviors mediated by olfactory perception. Megachile saussurei Radoszkowski (Hymenoptera, Megachilidae) is a principal insect pollinating alfalfa (Medicago sativa) in Northwestern China. The olfactory function have been less conducted, which provides a lot of possibilities for our research.

Results

Our results showed that 20 OBPs were identified in total. Multiple sequence alignment analysis indicated MsauOBPs were highly conserved with a 6-cysteine motif pattern and all belonged to the classic subfamily, coding 113-196 amino acids and sharing 41.32%-99.12% amino acid identity with known OBPs of other bees. Phylogenetic analysis indicated there were certain homologies existed among MsauOBPs and most sequences were clustered with that of Osmia cornuta (Hymenoptera, Megachilidae). Expression analysis showed the identified OBPs were mostly enriched in antennae instead of other four body parts, especially the MsauOBP2, MsauOBP3, MsauOBP4, MsauOBP8, MsauOBP11 and MsauOBP17, in which the MsauOBP2, MsauOBP4 and MsauOBP8 presented obvious tissue-biased expression pattern. Molecular docking results indicated MsauOBP4 might be the most significant protein in recognizing alfalfa flower volatile 3-Octanone, while MsauOBP13 might be the most crucial protein identifying (Z)-3-hexenyl acetate. It was also found the lysine was a momentous hydrophilic amino acid in docking simulations.

Conclusion

In this study, we identified and analyzed 20 OBPs of M. saussurei. The certain homology existed among these OBPs, while some degree of divergence could also be noticed, indicating the complex functions that different MsauOBPs performed. Besides, the M. saussurei and Osmia cornuta were very likely to share similar physiological functions as most of their OBPs were clustered together. MsauOBP4 might be the key protein in recognizing 3-Octanone, while MsauOBP13 might be the key protein in binding (Z)-3-hexenyl acetate. These two proteins might contribute to the alfalfa-locating during the pollination process. The relevant results may help determine the highly specific and effective attractants for M. saussurei in alfalfa pollination and reveal the molecular mechanism of odor-evoked pollinating behavior between these two species.

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Background

For a long period, insects have gradually adapted to the complex and ever-changing physiological environment with their sensitive olfactory system recognizing a large number of odor chemicals, which plays a crucial role in host selection, feeding, mating, and reproduction [1,2,3]. Insect’s antenna, covered by multi-type olfactory sensilla like the basiconic, coeloconic, and trichoid, is the central organ in sensing and recognizing external odors [4]. The sensilla are filled with potassium- and protein-rich fluid called sensillum lymph, which bathes the dendrites [5, 6]. Many chemosensation-related proteins secreted in sensillum lymph are involved in the complex olfactory-perception process, such as Odorant-binding proteins (OBPs), odorant receptors (ORs), chemosensory proteins (CSPs), ionotropic receptors (IRs), sensory neuron membrane proteins (SNMPs), and odorant-degrading enzymes (ODEs) [7, 8].

Among all those olfaction-related proteins, OBPs function as the initial step in odorant recognition and transduction [9, 10]. OBPs were a group of small, soluble, and acidic proteins with a highly-conserved structure [11, 12]. Generally, OBPs are classified into five diverse subtypes based on the number and model of conserved cysteines in their amino acid sequence [13], which includes Classical OBPs (those with 6 conserved cysteines), Minus-C OBPs (those with only 4 conserved cysteines), Plus-C OBPs (those with 8 conserved cysteines), dimer OBPs (those with 12 conserved cysteines) and Atypical OBPs (those with 9~10 conserved cysteines) [14, 15]. Upon encountering external chemical signals, such as pheromones, plant volatiles or odors from other species, odor molecules would enter the sensillum lymph through the massive pores on the sensilla, and OBPs in the lymph immediately recognize, bind and shift the newly-formed odor-OBP complexes to the ORs in sensory dendrites, which transform the chemical signals to electrophysiological signals and eventually trigger the corresponding behavior of insects [16,17,18].

OBPs have been intensively studied since the first report in a moth, Antheraea polyphemus [19]. Various OBPs and multiple functions accordingly have been identified. A class of GOBPs binding and transporting common odor molecules in the antennae of female Antheraea pernyi were identified [20] (Breer et al., 1990). Biochemical binding kinetics studies found the dual role of transporting and inactivating odorous substances [21, 22]. A study of Drosophila melanogaster mutants showed that OBPs are involved in the transport of odor molecules to ORs [23]. Besides, ApisOBP3 in Acyrthosiphon pisum [24], GmolGOBP2 in Grapholita molesta [25] and OBP6 in Meteorus pulchricornis [26] all demonstrated that OBPs could specifically recognize and screen specific chemical signals. Recently, The rapid development of techniques like electrophysiology, RNA interference, and gene knockout has directly revealed the necessity of OBPs for proper functioning in the olfactory system [27,28,29,30].

Information on three-dimensional structures and binding modes can elucidate the critical functions of the soluble olfactory proteins in insects’ daily behavior [31]. The interaction between OBPs and ligands based on molecular docking method has been widely conducted and the 3D-structures of over 20 OBPs in different insect species were reported including Diptera, Hemiptera, and Lepidoptera, etc. [2, 8]. Not only the OBPs, other soluble olfactory proteins such as CSPs were also studied using the molecular docking method. Previous studies even pointed out that molecular docking could function as a tool for screening the best ligands for insects [32]. These examples demonstrated that this virtual method has been reliable in the olfactory study of insects. To date, no such research has been conducted against MsauOBPs. Knowledge of the interaction of specific alfalfa flower volatile and MsauOBPs is still deficient.

M. saussurei is a principal pollinator of alfalfa (Medicago sativa L.) in Yumen area, Gansu province, which is one of the most important bases cultivating alfalfa in Northwest of China. Unlike the most intensively managed and studied alfalfa leaf-cutting bee (Megachile rotundata) [33] and other commercially produced bees, information in many aspects has been little known about this species. The objective of this study was to identify the odorant-binding proteins in female M. saussurei based on antenna-specific transcriptome analysis. Because males would die soon after they copulate with females [34], indicating female M. saussurei are the main force pollinating alfalfa. In this study, the antennae transcriptome sequencing of M. saussurei was performed and we also compared the putative OBPs in M. saussurei with those from other bees using phylogenetic analysis and determined the type of OBPs. The quantitative real-time PCR was thereafter applied to verify the expression pattern and level in five different tissues of M. saussurei. Finally, the interaction of MsauOBPs of two alfalfa flower volatiles was simulated using the molecular docking method. This is the first research investigating olfaction-related genes against M. saussurei, by which promising insights into the molecular mechanism of odor-evoked pollinating behavior and the development of highly specific and effective attractants for alfalfa pollination might be provided.

Results

Antenna transcriptome sequencing

In this research, three RNA-seq libraries were constructed, and a total of 45,573,892 raw reads were obtained in each sample. After removing reads containing over 5% unknown bases (971,132 in average), those containing adapters (1,579,565 on average) and those of low quality (40 on average), 43,023,155 clean reads on average were obtained, which accounts for 94.40% in total raw reads. The Q30 content in each sample was >90% (Table S3).

We obtained the unigenes with Trinity assembling followed by Tgicl-clustering in three samples. To improve the reliability of the assembly, Tgicl was used again to cluster the initial unigenes from three samples and finally generated the “all-unigene” assembly, which was used in the following analysis (Table S4). We acquired 77,444 unigenes in total with a total length of 184,461,623 bp and a mean length of 2381 bp. The values of N50, N70, and N90 were 4540 bp, 2,951 bp, and 1,263 bp respectively, and the GC content was 38.03% (Table S4). The number of unigene sequence sizes between 200-300 bp, 300-400 bp, and more than 3000 bp were 12041, 5779, and 22901 respectively, while the number of sequence sizes between 400-3000 bp was all lower than 5000 bp (Fig. S2). Results on assembly evaluation indicated only a small number of sequences were fragmented and missed in three samples and all-unigene, while more than 95% were able to match the sequences in the BUSCO database (Fig. S3), which indicated our unigenes were well assembled.

Functional annotation of unigenes

The 77,444 unigenes were functionally annotated in seven publicly accessed databases, among which 53,991 in NR (69.72%), 63,871 in NT (82.47%), 42,868 in SwissProt (55.35%), 42,052 in KOG (54.30%), 47,037 in KEGG (60.74%), 17,258 in GO (22.28%), and 43,002 in Pfam (55.53%) were successfully annotated, respectively (Table S5).

Figure 1 indicated that 72.20% of the M. saussurei unigenes annotated in NR database have best hits with genes in M. rotundata, followed by Osmia lignaria (5.88%) and Osmia bicornis (4.17%) (Fig. 1). Three functional categories, biological process, cellular component, and molecular function, were annotated in GO annotation (Fig. 2). In the biological process category, the genes expressed in the antennae were mostly enriched to the cellular process and metabolic process. In the molecular function category, binding and catalytic activity accounted for more than 80% of the total unigenes, while only two terms existed in the cellular component category, namely cellular anatomical entity and protein-containing complex. In KEGG analysis, unigenes were assigned and annotated into six different pathways, cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, and organismal systems. The specific term “signal transduction” with 7286 unigenes in the environmental information processing pathway was closely related to odorant binding proteins, having the second largest number of unigenes (Fig. 3).

Fig. 1
figure 1

Species classification of best blast hit in transcriptome analysis

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Fig. 2
figure 2

GO enrichment analysis of M. saussurei unigenes

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Fig. 3
figure 3

KEGG pathway analysis of M. saussurei unigenes

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Identification of odorant-binding protein genes

Based on the highly conserved amino acid sequence structure, we identified a total of 20 OBP genes from the M. saussurei antennae transcriptome and named them after MsauOBP1-5 and MsauOBP7-21. Detailed information on these OBPs is displayed in Table 1. All sequences contained complete open reading frame (ORF), coded 113-196 amino acids, and shared 41.32%-99.12% amino acid identity with known OBPs of other bees. Most OBPs contained a N-terminal signal peptide except MsauOBP1, 5, 7, 9, 12, 21. All sequences have been uploaded to GenBank, the accession number and other best blast match results can also be seen in Table 1.

Table 1 The sequence information of 20 identified odorant-binding proteins in M. saussurei antenna
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Multiple sequence alignment results indicated all putative OBPs displayed highly conserved amino acid sequence structure with six cysteine residues, which belonged to the Classic OBPs subfamily (Fig. 4), while other types of OBPs like Minus-C, Plus-C, Dimer, or Atypical OBPs were not found. The motif structure of MsauOBPs is (C1-X26-28-C2-X3-C3-X37-43-C4-X8-12-C5-X8-C6), where Xn stands for any n amino acids [9]. The expression level indicated MsauOBP2, 3, 4, 8, 11, and 17 were highly enriched in the M. saussurei antenna (Fig. 5B) and the relative expression level of 20 putative odorant-binding proteins in three biological samples of M. saussurei antenna was displayed in Fig. 5A.

Fig. 4
figure 4

Multiple-sequence alignment of 20 odorant-binding proteins in M. saussurei antenna. Red colour represents the six highly-conserved cysteines

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Fig. 5
figure 5

A Expression levels of 20 putative odorant-binding proteins from M. saussurei antenna (normalized by log10(FPKM+1)) in three samples (A1, A2, A3). B The actual FPKM values of M. saussurei OBPs

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To compare the homologous relationship of MsauOBPs with other species, the phylogenetic trees between the identified 20 putative M. saussurei OBPs and 119 known OBP protein sequences from other 24 species in Hymenoptera were then constructed (Fig. 6). The tree was divided into three branches, and all MsauOBPs were grouped in the same one. The putative MsauOBPs were most homologous to OcorOBPs of Osmia cornuta, among which MsauOBP8 and MsauOBP21 were clustered together with OcorOBP1; MsauOBP17, 19 and 20 were closed to OcorOBP5; MsauOBP4, 5, 13 and 14 were grouped with OcorOBP4; MsauOBP11 and MsauOBP12 were clustered with OcorOBP4; MsauOBP1 was grouped with OcorOBP6; MsauOBP2 and MsauOBP3 were grouped with OcorOBP3. While MsauOBP9 and MsauOBP10 were closed to AcerOBP10 of Apis cerana, MsauOBP7, 15, and 18 were closed to AmelOBP8 of Apis mellifera, MsauOBP16 was grouped with CgigGOBP56a of Colletes gigas.

Fig. 6
figure 6

Phylogenetic tree (bootstrap values ≥0.7 were shown) between MsauOBPs and OBPs from other 24 species. Acer: Apis cerana; Ador: Apis dorsata; Aflo: Apis florea; Alab: Apis laboriosa; Amel: Apis mellifera; Baff: Bombus affinis; Bbif: Bombus bifarius; Bhun: Bombus huntii; Bign: Bombus ignitus; Bimp: Bombus impatiens; Bpyr: Bombus pyrosoma; Bter: Bombus terrestris; Bvan: Bombus vancouverensis; Bvos: Bombus vosnesenskii; Ccal: Ceratina calcarata; Cgig: Colletes gigas; Hant: Hylaeus anthracinus; Hvol: Hylaeus volcanicus; Mrot: Megachile rotundata; Mgen: Megalopta genalis; Nmel: Nomia melanderi; Ocor: Osmia cornuta; Olig: Osmia lignaria; Obic: Osmia bicornis. Green strips represent MsauOBPs. The specific OBP and corresponding accession number were listed in Table S1

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Expression analysis of MsauOBPs based on quantitative real-time PCR

To further understand the expression level of 20 putative MsauOBPs, the quantitative real-time PCR experiment was conducted in different tissues. Results showed all MsauOBPs were differentially expressed in antennae and other body parts, and the expression variations were significant (Fig. 7). 10 out of 20 OBPs, including MsauOBP2, 3, 4, 7, 8, 10, 11, 13, 17, and 20, were highly expressed in antennae. While MsauOBP1, 5, 12, 14, 18, and 21 were highly expressed in heads. Only a small number of OBPs were expressed in legs and wings, among which some were even too low to be detected. MsauOBP9, 15, 16, and 19 had a lower expression in all tissues. MsauOBP2, MsauOBP8 and MsauOBP17 had a higher expression in antennae but extremely low expression levels in other body parts, implying their potential olfactory functions.

Fig. 7
figure 7

Relative expression levels (mean value ± SD) of M. saussurei OBPs in different tissues based on quantitative real-time PCR. Different lowercase letters indicate significant differences (one-way ANOVA followed by Tukey’s test, p < 0.05). AN, antennae; HE, head; LE, legs; WI, wing; AB, abdomen. The relative expression level of OBPs in M. saussurei abdomen was set to one

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Homologous modeling and molecular docking

Molecular docking results indicated the residues engaged in hydrogen bond (H-bond) formation varied significantly among MsauOBPs. In the docking results of MsauOBPs and 3-Octanone, LYS and LEU were the most frequently appeared residues in forming the H-bond (Table 2). All MsauOBPs successfully formed H-bond with 3-Octanone except for MsauOBP15. However, the binding sites of MsauOBP5, MsauOBP8, MsauOBP19, MsauOBP20, and MsauOBP21 were out of the potential domain of hydrophobic cavity. The MsauOBP1, MsauOBP10, and MsauOBP17 possessed two active binding sites, while the others only only had one (Fig. 8). The MsauOBP4 showed the best docking result with a mean binding energy of -20.84 kJ/mol. Similar to 3-Octanone, LYS was also repeatedly used in the docking of MsauOBP-(Z)-3-hexenyl acetate. Four OBPs, MsauOBP2, MsauOBP3, MsauOBP9, and MsauOBP16, showed no H-bond formation with (Z)-3-hexenyl acetate, which was greater than the number of OBP when docking with 3-Octanone (Table 2). The binding sites of MsauOBP8, MsauOBP14, and MsauOBP21 were out of the potential domain of hydrophobic cavity.Two active forming sites were detected in MsauOBP14 and 17, while three were found in MsauOBP13 with the lowest binding energy of -24.02 kJ/mol (Table 2, Fig. 8). Overall, more MsauOBPs tended to combine with 3-Octanone rather than (Z)-3-hexenyl acetate. However, among the MsauOBPs that could form H-bonds with both ligands, the mean binding energy in the docking of MsauOBP-(Z)-3-hexenyl acetate was generally lower than that of MsauOBP-3-Octanone.

Table 2 Molecular docking results of MsauOBPs with 3-Octanone and (Z)-3-hexenyl acetate
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Fig. 8
figure 8

Three-dimensional structures of interaction between M. saussurei and alfalfa flower volatile 3-Octanone and (Z)-3-hexenyl acetate based on molecular docking. A Molecular docking simulation of MsauOBP1/3-Oct, Z-3-HA; B, C Molecular docking simulation of MsauOBP2, MsauOBP3/3-Oct; D Molecular docking simulation of MsauOBP4/3-Oct, Z-3-HA; E Molecular docking simulation of MsauOBP5/Z-3-HA; F Molecular docking simulation of MsauOBP7/3-Oct, Z-3-HA; G Molecular docking simulation of MsauOBP9/3-Oct; H-K MsauOBP10-MsauOBP13/3-Oct, Z-3-HA; L Molecular docking simulation of MsauOBP14/3-Oct; M Molecular docking simulation of MsauOBP15/Z-3-HA; N Molecular docking simulation of MsauOBP16/3-Oct; O, P Molecular docking simulation of MsauOBP17, MsauOBP18/3-Oct, Z-3-HA; Q Molecular docking simulation of MsauOBP19/Z-3-HA; R Molecular docking simulation of MsauOBP20/Z-3-HA

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Discussion

Antennae are one of the most crucial sensory organs in insects, in which the chemosensory system contains multiple olfactory genes receiving chemical odors to mediate insect behaviors [35, 36]. Especially the odorant-binding proteins (OBPs), which were thought to be the first step in molecular recognition and the transport of semiochemicals [9]. To date, OBPs and their potential functions have been well-studied in many species, including Nasonia vitripennis [37], Leptinotarsa decemlineata [38], Cybister japonicus [39], Bemisia tabaci [40] (Zeng et al., 2019), Conogethes pinicolalis [41] and Meteorus pulchricornis [26] etc. M. saussurei was found to be an efficient pollinator of alfalfa in Northwestern China, and not much research was conducted against this species. To further understand the chemosensory information of M. saussurei, we conducted the antennal transcriptome to screen and identify the putative OBPs that might be involved in recognizing external odors and regulating their pollinating behaviors.

Based on our transcriptome results, 20 putative OBPs of M. saussurei were discovered, which contained 6 highly-conserved cysteines. The number was less than that of Nasonia vitripennis [37] (90 OBPs), similar to A. mellifera (21 OBPs) [42] and Cotesia vestalis (20 OBPs) [28], but more than M. rotundata (7 OBPs) [6], respectively. All putative OBPs belonged to the subgroup of Classic OBPs (Fig. 2) and encoded 143 amino acids on average, which was in a reasonable range compared with other species [28, 43, 44]. It has been pointed out that the number of OBPs could vary significantly across species [45]. In species where genome data has been published, for instance, Orthoptera, Locusta migratoria (22 OBPs); Hemiptera, Acyrthosiphon pisum (16 OBPs); Coleoptera, Tribolium castaneum (50 OBPs); Lepidoptera, Bombyx mori (45 OBPs); Hymenoptera, Apis mellifera (21 OBPs); M. rotundata (7 OBPs); Nasonia vitripennis (90 OBPs); Diptera, Drosophila melanogaster (52 OBPs) [6, 10]. It was hypothesized the diverse OBPs were probably related to the various semiochemicals in our environment [46], which formed the basis that different OBPs perform disparate functions. Besides, the real number of putative OBPs in M. saussurei could be more than 20, because a few OBPs were specifically expressed in other tissues of insects, such as gut [47], genital [48], legs and wings [49]. It’s highly possible that other OBPs were not included in our results, which might be one reason that we were not able to identify other types of OBPs.

The phylogenetic tree was often used to compare the homology relationship between different species. In this research, we collected 119 OBPs that were previously uploaded to GenBank from 24 species in Hymenoptera and constructed a phylogenetic tree with 20 predicated MsauOBPs. Results showed all MsauOBPs were clustered into the same branch, indicating there was certain homology existed. However, some differentiation could also be noticed, because not all sequences were grouped in the same clade. For instance, MsauOBP1 and MsauOBP16 were respectively clustered with other species, while other MsauOBPs were grouped in pairs or multi numbers (Fig. 4), which was similar to the phylogenetic results in other studies [28, 41, 44]. This divergence was probably due to the complex functions that different MsauOBPs performed. Previous studies even reported OBPs were extremely divergent in their sequences and identical amino acids between members of the same species, as well as between species, might be even lower than 10% [6]. Furthermore, our results also found that Osmia cornuta was the most homologous-closed species to M. saussurei in OBP sequences, which also belongs to the Megachilidae but in a different genus (Osmia), implying OBPs of these two species may share similar physiological functions.

Expression analysis with quantitative real-time PCR method indicated most putative MsauOBPs were highly expressed in antennae. Although a certain degree of expression could be seen in other tissues, such as MsauOBP5, MsauOBP13, MsauOBP18, and MsauOBP21 (Fig. 5), more MsauOBPs were presented as antennal-specific expression patterns, especially the MsauOBP2, MsauOBP8, and MsauOBP17, where the maximum differential expression occurred between antennae and other body parts, indicating the olfactory function these OBPs may possess. Similar results have been recorded in C. vestalis, in which the CvesOBP7, CvesOBP8, CvesOBP13, CvesOBP17, CvesOBP18, and CvesOBP19 were specifically enriched in female antennae, while CvesOBP9, and CvesOBP10 were significantly expressed in bodies [28]. In honey bees, AmelOBP9 and AmelOBP10 were reported to be highly expressed in non-olfactory tissues including brains, ovaries, and even eggs except many other antennal-specific enriched OBPs [42]. Besides, the SinvOBP10 of Solenopsis invicta, an imported fire ant, was also highly expressed in their brains at the pupal stage [50].

It has been reported the expression variation in different tissues probably corresponded to diverse physiological functions [51, 52]. For instance, the antennal-specific expressed AcerOBP1 can bind to the main components of the queen pheromones 9-ODA and 9-HDA (9-hydroxy-2(E)-decenoic acid) [53]. The leg-specific expressed AlinOBP11 of Adelphocoris lineolatus had important gustatory functions [54]. In some Lepidoptera insects, OBPs enriched in bodies may have the function of helping release the semiochemicals [41]. In this study, antennal-specific expressed OBPs, such as MsauOBP2, 3, 4, 8, 11, and 17, were highly possible to possess the olfactory function, which was similar to the fig wasp Wiebesia pumilae,, where this creature located its host Ficus pumila mainly through WpumOBP2 binding the decanal emitted by F. pumila [55]. Furthermore, the O.lotOBP6 of Odontothrips loti could strongly bind to p-Menth-8-en-2-one emitted by its host Medicago sativa and was the most crucial OBP in host-seeking [15]. Consequently, it’s reasonable to hypothesize that M. saussurei locate M. sativa through these highly expressed OBPs binding the single or multiple volatiles emitted by M. sativa to complete their feeding and pollination.

The interaction of MsauOBPs and two alfalfa flower volatiles 3-octanone and (Z)-3-hexenyl acetate was simulated by molecular docking method. Results showed most MsauOBPs could successfully bind with two ligands. It has been confirmed that the lower the binding energy, the better the binding effect [32]. In this study, MsauOBP4 showed the minimum value of binding energy when docking with 3-Octanone, while MsauOBP13 presented the lowest binding energy when docking with (Z)-3-hexenyl acetate. This implied MsauOBP4 and MsauOBP13 may play a crucial role in recognizing these two volatiles and may also contribute to the host location during the pollination process. Although more MsauOBPs tended to bind with 3-octanone, the mean binding energy of (Z)-3-hexenyl acetate was generally much lower, indicating that the combination between MsauOBPs and (Z)-3-hexenyl acetate was much more stable. Results also found the amino acid lysine appeared most frequently in docking simulations, which was also a momentous amino acid in other soluble olfactory proteins such as FoccOBP6 of Frankliniella occidentalis [8], OBP3 of Nilaparvata lugens [56] and MsepCSP14 of Mythimna separata [57]. It was found hydrophilic amino acids are more likely to form hydrogen bonds with ligands [58]. For instance, asparagine and serine in Hymenoptera [59], arginine, threonine, and aspartic acid in Lepidoptera [60, 61], glutamine in Hemiptera [62]. This was consistent with our result, in which lysine was also one of the hydrophilic amino acids.

Conclusions

In this study, we identified the OBPs, and conducted the phylogenetic and expression analysis. The interaction between two alfalfa flower volatiles and MsauOBPs was also simulated. Most OBPs were homologous while a certain degree of differences also existed. Six OBPs (MsauOBP2, 3, 4, 8, 11, and 17) mostly enriched in antennae were possibly involved in the olfactory functions. MsauOBP4 might be the key protein in recognizing 3-Octanone, while MsauOBP13 might be the key protein in binding (Z)-3-hexenyl acetate. These two proteins might contribute to the alfalfa-locating during the pollination process. The relevant results may help determine the highly specific and effective attractants for M. saussurei in alfalfa pollination and reveal the molecular mechanism of odor-evoked pollinating behavior between these two species. Further studies of these highly expressed OBPs using multi-methods are quite necessary, such as fluorescence binding assay, RNAi technique, and corresponding behavioral experiments, etc. Because these methods have been frequently used for the functional prediction and verification of insect OBPs. The relevant results may help determine the highly specific and effective attractants for M. saussurei in alfalfa pollination and reveal the molecular mechanism of odor-evoked pollinating behavior between these two species.

Methods

Antenna sample collection

The M. saussurei adults were captured in a blooming alfalfa field in the Yumen area (40◦45´N, 97◦36´E), Gansu province, China, in July 2022. To attract M. saussurei, the artificial foam nest (polystyrene bee board) was placed near the edges of the alfalfa field with the openings of the artificial nests facing the alfalfa field in a southeast direction [63, 64]. The size of artificial nests was maintained as instructed by Pitts-Singer and Bosch [65]. After M. saussurei was nested in these artificial nests, the emergence status and sex information of the adults were recorded every day. We carefully dissected the antennae from female M. saussurei in the laboratory and placed them in 1.5mL centrifugal tubes containing the RNA later buffer solution (Invitrogen, Carlsbad, CA, USA) [18]. The tubes were preserved at -80℃ until RNA extraction.

RNA extraction and transcriptome sequencing

Fifty pairs of antennae from M. saussurei adult females were used for total RNA extraction using TRIzol Reagent (Invitrogen, Waltham, MA, USA) following the manufacturer’s standard protocols (50 pairs antennae formed one sample, three samples (A1, A2, and A3) were set in total). The concentration and quality of RNA were verified using Fragment Analyzer 5200 (Agilent Technologies, Palo Alto, Canada). The cDNA library construction and transcriptome sequencing were performed on the DNBSEQ-500 platform at Wuhan BGI Technology (Wuhan, China) and a detailed flowchart was displayed in Fig. S1.

De novo assembly and functional annotation

To ensure the data reliability, we obtained clean reads from raw reads by filtering and deleting those reads of low quality, containing adapters and over 5% unknown bases. The clean reads were then assembled with Trinity v2.0.6 (https://github.com/trinityrnaseq/trinityrnaseq/wiki) using default parameters [66]. Then the unigenes from the three samples were pooled together to form the “all-unigene” by clustering reads and removing redundancy with the TGI Clustering Tool (TGICL) [67]. The quality of the assembled transcripts (unigenes) was thereafter evaluated using the BUSCO (Benchmarking Universal Single-Copy Orthologs) (https://busco.ezlab.org/), and the integrity of the transcriptome assembly was illustrated by comparison with conserved genes.

The coding sequence (CDS) in unigenes was identified using TransDecoder software by first extracting the longest open reading frame, and then Blast comparison against the Pfam protein homologous sequences in the SwissProt database and Hmmscan search to predict the coding regions. The unigenes were annotated against seven publicly accessed databases, the Kyoto Encyclopedia of Genes and Genomes (KEGG), the Gene Ontology (GO), the Non-redundant Protein Sequence Database (NR), Nucleotide Sequence Database (NT), the Protein Families Database (Pfam), Swiss-prot protein sequence database (Swiss-prot) and clusters of orthologous groups for eukaryotic complete genomes (KOG) with a threshold E-value < 1e-5. The expression level of each unigene was calculated by RSEM software (RNA-Seq by Expectation Maximization) with default parameters and presented as FPKM (fragments per kilobase of transcript per million mapped fragments) values.

Identification of odorant-binding protein genes and phylogenetic analysis

Candidate unigenes encoding putative odorant-binding proteins (OBPs) were selected from the assembly results. They were manually checked by performing a BLASTx search against the NR database with a threshold E-value < 1e-5 [68]. The open reading frame (ORF) of candidate OBP genes was predicted by NCBI ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder). The N-terminal signal peptides were predicted by Signal P4.0 (http://www.cbs.dtu.dk/services/SignalP/).

We applied multiple amino acid sequence alignment with MUSCLE and constructed phylogenetic trees of putative OBP genes using the neighbor-joining (NJ) method with default parameters in MEGA v11.0 software. The reliability of the tree structure and node support was assessed using a bootstrap method with 1000 replicates and the phylogenetic tree was visualized in the Interactive Tree of Life (iTOL) (https://itol.embl.de/). Sequences of OBP genes from other bees were searched and selected from NCBI and used in the phylogenetic tree construction (Table S1). We finally aligned putative OBPs using GenDoc software and determined the type of putative OBPs.

Expression analysis by quantitative real-time PCR

After we identified the OBPs from the antennal transcriptome, we verified their expression levels in different tissues of M. saussurei using the quantitative real-time PCR method (RT-qPCR). Antenna, heads, legs, wings, and abdomen from 20 individuals were respectively collected and pooled together as one sample. Total RNA was extracted with TRIzol reagent (Invitrogen) and the cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan). The total volume of the PCR reaction system was 25μl, which contains 12.5 µl of SYBR Premix Ex TaqTM, 0.5 µl of forward primer, 0.5 µl of reverse primer, 2 μl of sample cDNA and 8.5 μl of double-distilled H2O. This PCR system was performed under the conditions of 95℃ for 30 s; 40 cycles of 95℃ for 5 s and 60℃ for 30 s; 65℃ to 95℃ in increments of 0.5℃ for 5 s. Negative controls with ddH2O were included. Gene-specific primers (Table S2) were designed using the Primer 3.0 plus server in NCBI. Nuclear β-actin was used as the internal reference gene and abdomen samples were used as the control group. Three biological replicates and three technical replicates were applied for each experiment.

The relative expression level of OBP genes was normalized using the comparative 2−∆∆Ct method [69]. One-way ANOVA analysis was applied to compare the expression levels between tissues, followed by Tukey’s post hoc comparison test for the significant differences. The data analysis and plot-making were both conducted using GraphPad Prism 9.0 software.

Homologous modeling and molecular docking

The online platform SWISS-MODEL (https://swissmodel.expasy.org) was used to predict the three-dimensional structure of all MsauOBPs. Models with similarity >30% were selected as reference templates. The PROCHECK program [70] was used to assess the generated MsauOBP models. 3-Octanone and (Z)-3-hexenyl acetate are two main components of alfalfa flower volatiles with relatively high content [71,72,73,74]. Ligand molecules were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov). The Autodock 4.2.6 and AutoDock Tools 1.5.7 with default parameters were used to conduct the molecular docking between MsauOBPs and two ligands. The docking results were visualized by PYMOL software.

Availability of data and materials

All data support this research is included in this article and supplementary file. The original reads of transcriptome sequencing from this study were uploaded to NCBI Sequence Read Archive with accession number PRJNA977226. The sequences of 20 MsauOBPs were also submitted to Genbank with accession number OR266110-OR266114, OR266116-OR266130. The internal reference gene, Nuclear β-actin, was obtained from transcriptome sequencing data with Genbank accession number OR405375.

Abbreviations

OBPs:

Odorant binding proteins

MsauOBPs:

Megachile saussurei odorant binding proteins

ORs:

odorant receptors

CSPs:

chemosensory proteins

IRs:

ionotropic receptors

SNMPs:

sensory neuron membrane proteins

ODEs:

odorant-degrading enzymes

TGICL:

TIGR Gene Indices clustering tools

BUSCO:

Benchmarking Universal Single-Copy Orthologs

CDS:

coding sequence

ORFs:

Open reading frame

KEGG:

Kyoto Encyclopedia of Genes and Genomes

GO:

Gene ontology

NR:

Non-redundant protein sequence database

NT:

Nucleotide Sequence Database

Pfam:

Protein families database

Swiss-prot:

Swiss-prot protein sequence database

KOG:

clusters of orthologous groups for eukaryotic complete genomes

RSEM:

RNA-Seq by Expectation Maximization

FPKM:

Fragments per kilobase per million reads

RT-qPCR:

Fluorescent quantitative real-time PCR

References

  1. Bruce TJ, Wadhams LJ, Woodcock CM. Insect host location: a volatile situation. Trends Plant Sci. 2005;10(6):269–74. https://doi.org/10.1016/j.tplants.2005.04.003.

    Article  CAS  PubMed  Google Scholar 

  2. Brito NF, Moreira MF, Melo AC. A look inside odorant-binding proteins in insect chemoreception. J Insect Physiol. 2016;95:51–65. https://doi.org/10.1016/j.jinsphys.2016.09.008.

    Article  CAS  PubMed  Google Scholar 

  3. Cui XN, Liu DG, Sun KK, He Y, Shi XQ. Expression profiles and functional characterization of two odorant-binding proteins from the apple buprestid beetle Agrilus mali (Coleoptera: Buprestidae). J Econ Entomol. 2018;111(3):1420–32. https://doi.org/10.1093/jee/toy066.

    Article  PubMed  Google Scholar 

  4. Pelosi P, Zhou JJ, Ban LP, Calvello M. Soluble proteins in insect chemical communication. Cell Mol Life Sci. 2006;63(14):1658–76. https://doi.org/10.1007/s00018-005-5607-0.

    Article  CAS  PubMed  Google Scholar 

  5. Smith DP. Odor and pheromone detection in Drosophila melanogaster. Pflugers Arch. 2007;454(5):749–58. https://doi.org/10.1007/s00424-006-0190-2.

    Article  CAS  PubMed  Google Scholar 

  6. Pelosi P, Iovinella I, Felicioli A, Dani FR. Soluble proteins of chemical communication: an overview across arthropods. Front Physiol. 2014;5:320. https://doi.org/10.3389/fphys.2014.00320.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Leal WS. Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes. Annu Rev Entomol. 2013;58:373–91. https://doi.org/10.1146/annurev-ento-120811-153635.

    Article  CAS  PubMed  Google Scholar 

  8. Li X, Cheng J, Chen L, Huang J, Zhang Z, Zhang J, Ren X, Hafeez M, Zhou S, Dong W, Lu Y. Comparison and functional analysis of odorant-binding proteins and chemosensory proteins in two closely related thrips species, Frankliniella occidentalis and Frankliniella intonsa (Thysanoptera: Thripidae) based on antennal transcriptome analysis. Int J Mol Sci. 2022;23(22):13900. https://doi.org/10.3390/ijms232213900.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhou JJ, Huang W, Zhang GA, Pickett JA, Field LM. “Plus-C” odorant-binding protein genes in two Drosophila species and the malaria mosquito Anopheles gambiae. Gene. 2004;327(1):117–29. https://doi.org/10.1016/j.gene.2003.11.007.

    Article  CAS  PubMed  Google Scholar 

  10. Li JB, Yin MZ, Yao WC, Ma S, Dewer Y, Liu XZ, Wang YY, Wang CW, Li BP, Zhu XY. Genome-wide analysis of odorant-binding proteins and chemosensory proteins in the bean bug Riptortus pedestris. Front Physiol. 2022;13:949607. https://doi.org/10.3389/fphys.2022.949607.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Ahmed T, Zhang T, Wang Z, He K, Bai S. Molecular cloning, expression profile, odorant affinity, and stability of two odorant-binding proteins in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae). Arch Insect Biochem Physiol. 2017;94(2):e21374. https://doi.org/10.1002/arch.21374.

    Article  CAS  Google Scholar 

  12. Suzuki RH, Hanada T, Hayashi Y, Shigenobu S, Maekawa K, Hojo MK. Gene expression profiles of chemosensory genes of termite soldier and worker antennae. Insect Mol Biol. 2023;32(4):424–35. https://doi.org/10.1111/imb.12841.

    Article  CAS  PubMed  Google Scholar 

  13. Zhou JJ, He XL, Pickett JA, Field LM. Identification of odorant-binding proteins of the yellow fever mosquito Aedes aegypti: genome annotation and comparative analyses. Insect Mol Biol. 2008;17(2):147–63. https://doi.org/10.1111/j.1365-2583.2007.00789.x.

    Article  CAS  PubMed  Google Scholar 

  14. Spinelli S, Lagarde A, Iovinella I, Legrand P, Tegoni M, Pelosi P, Cambillau C. Crystal structure of Apis mellifera OBP14, a C-minus odorant-binding protein, and its complexes with odorant molecules. Insect Biochem Mol Biol. 2012;42(1):41–50. https://doi.org/10.1016/j.ibmb.2011.10.005.

    Article  CAS  PubMed  Google Scholar 

  15. Liu Y, Luo Y, Du L, Ban L. Antennal transcriptome analysis of olfactory genes and characterization of odorant binding proteins in Odontothrips loti (Thysanoptera: Thripidae). Int J Mol Sci. 2023;24(6):5284. https://doi.org/10.3390/ijms24065284.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhou JJ, Vieira FG, He XL, Smadja C, Liu R, Rozas J, Field LM. Genome annotation and comparative analyses of the odorant-binding proteins and chemosensory proteins in the pea aphid Acyrthosiphon pisum. Insect Mol Biol. 2010;19(Suppl 2):113–22. https://doi.org/10.1111/j.1365-2583.2009.00919.x.

    Article  CAS  PubMed  Google Scholar 

  17. Venthur H, Zhou JJ. Odorant receptors and odorant-binding proteins as insect pest control targets: a comparative analysis. Front Physiol. 2018;9:1163. https://doi.org/10.3389/fphys.2018.01163.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Guo B, Hao E, Qiao H, Wang J, Wu W, Zhou J, Lu P. Antennal transcriptome analysis of olfactory genes and characterizations of odorant binding proteins in two woodwasps, Sirex noctilio and Sirex nitobei (Hymenoptera: Siricidae). BMC Genomics. 2021;22(1):172. https://doi.org/10.1186/s12864-021-07452-1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Vogt RG, Riddiford LM. Pheromone binding and inactivation by moth antennae. Nature. 1981;1981(293):161–3. https://doi.org/10.1038/293161a0.

    Article  Google Scholar 

  20. Breer H, Krieger J, Raming K. A novel class of binding proteins in the antennae of the silk moth Antheraea pernyi. Insect Biochem. 1990;20(7):735–40. https://doi.org/10.1016/0020-1790(90)90088-C.

    Article  CAS  Google Scholar 

  21. Vogt RG, Rybczynski R, Lerner MR. Molecular cloning and sequencing of general odorant-binding proteins GOBP1 and GOBP2 from the tobacco hawk moth Manduca sexta: comparisons with other insect OBPs and their signal peptides. J Neurosci. 1991;11(10):2972–84. https://doi.org/10.1523/JNEUROSCI.11-10-02972.1991.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ziegelberger G. Redox-shift of the pheromone-binding protein in the silkmoth Antheraea polyphemus. Eur J Biochem. 1995;232(3):706–11. https://doi.org/10.1111/j.1432-1033.1995.0706a.x.

    Article  CAS  PubMed  Google Scholar 

  23. Kim MS, Repp A, Smith DP. LUSH odorant-binding protein mediates chemosensory responses to alcohols in Drosophila melanogaster. Genetics. 1998;150(2):711–21. https://doi.org/10.1093/genetics/150.2.711.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Qiao H, Tuccori E, He X, Gazzano A, Field L, Zhou JJ, Pelosi P. Discrimination of alarm pheromone (E)-beta-farnesene by aphid odorant-binding proteins. Insect Biochem Mol Biol. 2009;39(5–6):414–9. https://doi.org/10.1016/j.ibmb.2009.03.004.

    Article  CAS  PubMed  Google Scholar 

  25. Li G, Chen X, Li B, Zhang G, Li Y, Wu J. Binding properties of general odorant binding proteins from the oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae). PLoS One. 2016;11(5):e0155096. https://doi.org/10.1371/journal.pone.0155096.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Li YJ, Hong TL, Chen HC, Gu FM, Liu ZX, You S, Wu FA, Sheng S, Wang J. Odorant-binding protein 6 contributes high binding affinity to insecticides in a parasitic wasp Meteorus pulchricornis (Hymenoptera: Braconidae). J Agric Food Chem. 2023;71(11):4498–509. https://doi.org/10.1021/acs.jafc.2c08390.

    Article  CAS  PubMed  Google Scholar 

  27. Li HL, Song XM, Wu F, Qiu YL, Fu XB, Zhang LY, Tan J. Chemical structure of semiochemicals and key binding sites together determine the olfactory functional modes of odorant-binding protein 2 in Eastern honey bee, Apis cerana. Int J Biol Macromol. 2020;145:876–84. https://doi.org/10.1016/j.ijbiomac.2019.11.189.

    Article  CAS  PubMed  Google Scholar 

  28. Zhou YN, Xie S, Chen JN, Wang ZH, Yang P, Zhou SC, Pang L, Li F, Shi M, Huang JH, Chen XX. Expression and functional characterization of odorant-binding protein genes in the endoparasitic wasp Cotesia vestalis. Insect Sci. 2021;5:1354–68. https://doi.org/10.1111/1744-7917.12861.

    Article  CAS  Google Scholar 

  29. Wu Y, Li Y, Chu W, Niu T, Feng X, Ma R, Liu H. Expression and functional characterization of odorant-binding protein 2 in the predatory mite Neoseiulus barkeri. Insect Sci. 2023;30(5):1493–506. https://doi.org/10.1111/1744-7917.13156.

    Article  CAS  PubMed  Google Scholar 

  30. Liggri PGV, Tsitsanou KE, Stamati ECV, Saitta F, Drakou CE, Leonidas DD, Fessas D, Zographos SE. The structure of AgamOBP5 in complex with the natural insect repellents Carvacrol and Thymol: Crystallographic, fluorescence and thermodynamic binding studies. Int J Biol Macromol. 2023;237:124009. https://doi.org/10.1016/j.ijbiomac.2023.124009.

    Article  CAS  PubMed  Google Scholar 

  31. Tomaselli S, Crescenzi O, Sanfelice D, Eiso AB, Wechselberger R, Angeli S, Scaloni A, Boelens R, Tancredi T, Pelosi P, Picone D. Solution structure of a chemosensory protein from the desert locust Schistocerca gregaria. Biochemistry. 2006;45:10606–13. https://doi.org/10.1021/bi060998w.

    Article  CAS  PubMed  Google Scholar 

  32. Venthur H, Mutis A, Zhou J-J, Quiroz A. Ligand binding and homology modelling of insect odorant-binding proteins. Physiol Entomol. 2014;39:183–98. https://doi.org/10.1111/phen.12066.

    Article  CAS  Google Scholar 

  33. Pitts-Singer TL, Cane JH. The alfalfa leafcutting bee, Megachile rotundata: the world’s most intensively managed solitary bee. Annu Rev Entomol. 2011;56:221–37. https://doi.org/10.1146/annurev-ento-120709-144836.

    Article  CAS  PubMed  Google Scholar 

  34. Eickwort GC, Ginsberg HS. Foraging and mating behavior in Apoidea. Annu Rev Entomol. 1980;1980(25):421–46. https://doi.org/10.1146/annurev.en.25.010180.002225.

    Article  Google Scholar 

  35. Fan J, Francis F, Liu Y, Chen JL, Cheng DF. An overview of odorant-binding protein functions in insect peripheral olfactory reception. Genet Mol Res. 2011;10(4):3056–69. https://doi.org/10.4238/2011.

    Article  CAS  PubMed  Google Scholar 

  36. Sims C, Birkett MA, Withall DM. Enantiomeric discrimination in insects: the role of OBPs and ORs. Insects. 2022;13(4):368. https://doi.org/10.3390/insects13040368.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Vieira FG, Forêt S, He X, Rozas J, Field LM, Zhou JJ. Unique features of odorant-binding proteins of the parasitoid wasp Nasonia vitripennis revealed by genome annotation and comparative analyses. PLoS One. 2012;7(8):e43034. https://doi.org/10.1371/journal.pone.0043034.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Liu Y, Sun L, Cao D, Walker WB, Zhang Y, Wang G. Identification of candidate olfactory genes in Leptinotarsa decemlineata by antennal transcriptome analysis. Front Ecol Evol. 2015;3:60. https://doi.org/10.3389/fevo.2015.00060.

    Article  Google Scholar 

  39. Song LM, Jiang X, Wang XM, Li JD, Zhu F, Tu XB, Zhang ZH, Ban LP. Male tarsi specific odorant-binding proteins in the diving beetle Cybister japonicus sharp. Sci Rep. 2016;6:31848. https://doi.org/10.1038/srep31848.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Zeng Y, Yang YT, Wu QJ, Wang SL, Xie W, Zhang YJ. Genome-wide analysis of odorant-binding proteins and chemosensory proteins in the sweet potato whitefly, Bemisia tabaci. Insect Sci. 2019;26:620–34. https://doi.org/10.1111/1744-7917.12576.

    Article  CAS  PubMed  Google Scholar 

  41. Jing D, Zhang T, Prabu S, Bai S, He K, Wang Z. Molecular characterization and volatile binding properties of pheromone binding proteins and general odorant binding proteins in Conogethes pinicolalis (Lepidoptera: Crambidae). Int J Biol Macromol. 2020;146:263–72. https://doi.org/10.1016/j.ijbiomac.2019.12.248.

    Article  CAS  PubMed  Google Scholar 

  42. Forêt S, Maleszka R. Function and evolution of a gene family encoding odorant binding-like proteins in a social insect, the honey bee (Apis mellifera). Genome Res. 2006;16(11):1404–13. https://doi.org/10.1101/gr.5075706.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Zhou JJ. Odorant-binding proteins in insects. Vitam Horm. 2010;83:241–72. https://doi.org/10.1016/S0083-6729(10)83010-9.

    Article  CAS  PubMed  Google Scholar 

  44. He H, Crabbe MJC, Ren Z. Genome-wide identification and characterization of the chemosensory relative protein genes in Rhus gall aphid Schlechtendalia chinensis. BMC Genomics. 2023;24(1):222. https://doi.org/10.1186/s12864-023-09322-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Rihani K, Ferveur J-F, Briand L. The 40-year mystery of insect odorant-binding proteins. Biomolecules. 2021;11:509. https://doi.org/10.3390/biom11040509.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Pelosi P, Calvello M, Ban L. Diversity of odorant-binding proteins and chemosensory proteins in insects. Chem Senses. 2005;30(Suppl 1):i291-2. https://doi.org/10.1093/chemse/bjh229.

    Article  CAS  PubMed  Google Scholar 

  47. Ribeiro JM, Genta FA, Sorgine MH, Logullo R, Mesquita RD, Paiva-Silva GO, Majerowicz D, Medeiros M, Koerich L, Terra WR, Ferreira C, Pimentel AC, Bisch PM, Leite DC, Diniz MM, da SGV Junior JL, Da Silva ML, Araujo RN, Gandara AC, Brosson S, Salmon D, Bousbata S, González-Caballero N, Silber AM, Alves-Bezerra M, Gondim KC, Silva-Neto MA, Atella GC, Araujo H, Dias FA, Polycarpo C, Vionette-Amaral RJ, Fampa P, Melo AC, Tanaka AS, Balczun C, Oliveira JH, Gonçalves RL, Lazoski C, Rivera-Pomar R, Diambra L, Schaub GA, Garcia ES, Azambuja P, Braz GR, Oliveira PL. An insight into the transcriptome of the digestive tract of the bloodsucking bug, Rhodnius prolixus. PLoS Negl Trop Dis. 2014;8(1):e2594. https://doi.org/10.1371/journal.pntd.0002594.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Sun YL, Huang LQ, Pelosi P, Wang CZ. Expression in antennae and reproductive organs suggests a dual role of an odorant-binding protein in two sibling Helicoverpa species. PLoS One. 2012;7(1):e30040. https://doi.org/10.1371/journal.pone.0030040.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Pelosi P, Iovinella I, Zhu J, Wang G, Dani FR. Beyond chemoreception: diverse tasks of soluble olfactory proteins in insects. Biol Rev Camb Philos Soc. 2018;93(1):184–200. https://doi.org/10.1111/brv.12339.

    Article  PubMed  Google Scholar 

  50. Zhang W, Wanchoo A, Ortiz-Urquiza A, Xia Y, Keyhani NO. Tissue, developmental, and caste-specific expression of odorant binding proteins in a eusocial insect, the red imported fire ant, Solenopsis invicta. Sci Rep. 2016;6:35452. https://doi.org/10.1038/srep35452.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. He P, Zhang J, Liu NY, Zhang YN, Yang K, Dong SL. Distinct expression profiles and different functions of odorant binding proteins in Nilaparvata lugens Stål. PLoS One. 2011;6(12):e28921. https://doi.org/10.1371/journal.pone.0028921.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Yue Y, Ma C, Zhang Y, Chen HS, Guo JY, Liu TH, Zhou ZS. Characterization and functional analysis of OcomOBP7 in Ophraella communa Lesage. Insects. 2023;14(2):190. https://doi.org/10.3390/insects14020190.

    Article  PubMed  PubMed Central  Google Scholar 

  53. Du H, Su W, Huang J, Ding G. Sex-biased expression of olfaction-related genes in the antennae of Apis cerana (Hymenoptera: Apidae). Genes (Basel). 2022;13(10):1771. https://doi.org/10.3390/genes13101771.

    Article  CAS  PubMed  Google Scholar 

  54. Zhao HT, Zhao WM, Gao PF, Zhang GX, Jiang YS. Sequence and expression characterization of an OBP1 gene in the Asian honeybee, Apis cerana cerana (Hymenoptera: Apidae). Appl Entomol Zool. 2014;49:189–96. https://doi.org/10.1007/s13355-013-0228-9.

    Article  CAS  Google Scholar 

  55. Wang R, Yang Y, Jing Y, Segar ST, Zhang Y, Wang G, Chen J, Liu QF, Chen S, Chen Y, Cruaud A, Ding YY, Dunn DW, Gao Q, Gilmartin PM, Jiang K, Kjellberg F, Li HQ, Li YY, Liu JQ, Liu M, Machado CA, Ming R, Rasplus JY, Tong X, Wen P, Yang HM, Yang JJ, Yin Y, Zhang XT, Zhang YY, Yu H, Yue Z, Compton SG, Chen XY. Molecular mechanisms of mutualistic and antagonistic interactions in a plant-pollinator association. Nat Ecol Evol. 2021;5(7):974–86. https://doi.org/10.1038/s41559-021-01469-1.

    Article  PubMed  Google Scholar 

  56. Zhang J, Mao K, Ren Z, Jin R, Zhang Y, Cai T, He S, Li J, Wan H. Odorant binding protein 3 is associated with nitenpyram and sulfoxaflor resistance in Nilaparvata lugens. Int J Biol Macromol. 2022;209:1352–8. https://doi.org/10.1016/j.ijbiomac.2022.04.100.

    Article  CAS  PubMed  Google Scholar 

  57. Younas A, Waris MI, Shaaban M, Tahir UlQ M, Wang MQ. Appraisal of MsepCSP14 for chemosensory functions in Mythimna separata. Insect Sci. 2022;29(1):162–76. https://doi.org/10.1111/1744-7917.12909.

    Article  CAS  PubMed  Google Scholar 

  58. Cui Z, Liu Y, Wang G, Zhou Q. Identification and functional analysis of a chemosensory protein from Bactrocera minax (Diptera: Tephritidae). Pest Manag Sci. 2022;78(8):3479–88. https://doi.org/10.1002/ps.6988.

    Article  CAS  PubMed  Google Scholar 

  59. Li HL, Ni CX, Tan J, Zhang LY, Hu FL. Chemosensory proteins of the eastern honeybee, Apis cerana: identification, tissue distribution and olfactory related functional characterization. Comp Biochem Physiol B Biochem Mol Biol. 2016;194–195:11–9. https://doi.org/10.1016/j.cbpb.2015.11.014.

    Article  CAS  PubMed  Google Scholar 

  60. Li G, Chen X, Chen L, Wang WQ, Wu JX. Functional analysis of the chemosensory protein GmolCSP8 from the oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae). Front Physiol. 2019;10:00552.

    Article  Google Scholar 

  61. Singh S, Tyagi C, Rather IA, Sabir JSM, Hassan MI, Singh A, Singh IK. Molecular modeling of chemosensory protein 3 from Spodoptera litura and its binding property with plant defensive metabolites. Int J Mol Sci. 2020;21:4073. https://doi.org/10.3390/ijms21114073.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Peng X, Liu L, Huang YX, Wang SJ, Li DX, Chen ST, et al. Involvement of chemosensory proteins in host plant searching in the bird cherryoat aphid. Insect Sci. 2020;28:1338–53. https://doi.org/10.1111/1744-7917.12865.

    Article  CAS  PubMed  Google Scholar 

  63. Fauria K, Campan R, Grimal A. Visual marks learned by the solitary bee Megachile rotundata for localizing its nest. Anim Behav. 2004;67:523–30. https://doi.org/10.1016/j.anbehav.2003.06.002.

    Article  Google Scholar 

  64. Pitts-Singer TL. Intended release and actual retention of alfalfa leafcutting bees (Hymenoptera: Megachilidae) for pollination in commercial alfalfa seed fields. J Econ Entomol. 2013;106(2):576–86. https://doi.org/10.1603/ec12416.

    Article  PubMed  Google Scholar 

  65. Pitts-Singer TL, Bosch J. Nest establishment, pollination efficiency, and reproductive success of Megachile rotundata (Hymenoptera: Megachilidae) in relation to resource availability in field enclosures. Environ Entomol. 2010;39(1):149–58. https://doi.org/10.1603/EN09077.

    Article  PubMed  Google Scholar 

  66. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29:644–52. https://doi.org/10.1038/nbt.1883.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Pertea G, Huang X, Liang F, Antonescu V, Sultana R, Karamycheva S, et al. TIGR gene indices clustering tools (TGICL): a software system for fast clustering of large EST datasets. Bioinformatics. 2003;19:651–2. https://doi.org/10.1093/bioinformatics/btg034.

    Article  CAS  PubMed  Google Scholar 

  68. Liu Y, Du L, Zhu Y, Yang S, Zhou Q, Wang G, Liu Y. Identification and sex-biased profiles of candidate olfactory genes in the antennal transcriptome of the parasitoid wasp Cotesia vestalis. Comp Biochem Physiol Part D Genomics Proteomics. 2020;34:100657. https://doi.org/10.1016/j.cbd.2020.100657.

    Article  CAS  PubMed  Google Scholar 

  69. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method. Methods. 2001;25:402–8. https://doi.org/10.1006/METH.2001.1262.

    Article  CAS  PubMed  Google Scholar 

  70. Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: A program to check the stereochemical quality of protein structures. J Appl Crystallogr. 1993;26:283–91. https://doi.org/10.1107/S0021889892009944.

    Article  CAS  Google Scholar 

  71. Yang DS, Lei Z, Bedair M, Sumner LW. An optimized SPME-GC-MS method for volatile metabolite profiling of different Alfalfa (Medicago sativa L.) Tissues. Molecules. 2021;26(21):6473. https://doi.org/10.3390/molecules26216473.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Light DM, Kamm JA, Buttery RG. Electroantennogram response of alfalfa seed chalcid, Bruchophagus roddi (Hymenoptera: Eurytomidae) to host- and nonhost-plant volatiles. J Chem Ecol. 1992;18(3):333–52. https://doi.org/10.1007/BF00994235.

    Article  CAS  PubMed  Google Scholar 

  73. Loper GM, Lapioli AM. Photoperiodic effects on the emanation of volatiles from Alfalfa (Medicago sativa L.) florets. Plant Physiol. 1972;49(5):729–32. https://doi.org/10.1104/pp.49.5.729.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Blackmer JL, Rodriguez-Saona C, Byers JA, Shope KL, Smith JP. Behavioral response of Lygus hesperus to conspecifics and headspace volatiles of alfalfa in a Y-tube olfactometer. J Chem Ecol. 2004;30(8):1547–64. https://doi.org/10.1023/b:joec.0000042067.27698.30.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Yumen Fenghua grass industry Co., LTD for their technical guidance and support in collecting M. saussurei samples.

Funding

This research was supported by the Strategic Research and Consulting Project of Chinese Academy of Engineering-Evaluation, Screening, Functional identification and utilization of important grass germplasm resources in arid cold region of Gansu Province (No. 2021-DFZD-21-4), National Key Research and Development Program: Inter-Governmental Science and Technology Innovation Program (No.2022YFE0115200) and the Fund for Disciplinary key team construction for Agricultural insect and pest control of College of Plant Protection, Gansu Agricultural University (No. GSAU-XKJS-2023).

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Authors and Affiliations

  1. Key Laboratory of Grassland Ecosystem of Ministry of Education, and Sino-U.S. Centers for Grazingland Ecosystem Sustainability, College of Grassland Science, Gansu Agricultural University, Lanzhou, 730070, China

    Wei-Zhen Li, Wen-Juan Kang & Shang-Li Shi

  2. College of Plant Protection, Biocontrol Engineering Laboratory of Crop Diseases and Pests of Gansu Province, Gansu Agricultural University, Lanzhou, 730070, China

    Jing-Jiang Zhou & Su-Qin Shang

  3. Department of Biological Chemistry, Rothamsted Research, Harpenden, Hertfordshire, UK

    Jing-Jiang Zhou

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  1. Wei-Zhen Li
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  2. Wen-Juan Kang
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  3. Jing-Jiang Zhou
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  4. Su-Qin Shang
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Contributions

LWZ, SSQ and SSL designed this study together. LWZ collected samples, performed the studies, carried out the bioanalysis, RT-qPCR and drafted the manuscript. KWJ participated in performing the experiments and data analysis. SSQ, SSL and ZJJ revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Su-Qin Shang or Shang-Li Shi.

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Supplementary Information

Additional file 1: Figure S1.

Flow chart of mRNA library construction. Figure S2. The distribution in sequence size of all unigenes. Figure S3. The assembly evaluation based on BUSCO. Complete: Sequences that matched to the records of the BUSCO database; F(fragmented): Partial sequences that matched to the records of the BUSCO database; D(duplicate): Multiple genes matched to one record of the BUSCO database; M(missing): Sequences that were filtered out. Table S1. OBP genes information of other species in phylogenetic analysis. Table S2. Gene-specific primers used for quantitative real-time PCR. Table S3. Quality statistics of filtered Reads in transcriptome sequencing. Table S4. The quality indicators of unigenes after Denovo assembly. Table S5. Functional annotation results of unigenes.

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Li, WZ., Kang, WJ., Zhou, JJ. et al. The antennal transcriptome analysis and characterizations of odorant-binding proteins in Megachile saussurei (Hymenoptera, Megachilidae). BMC Genomics 24, 781 (2023). https://doi.org/10.1186/s12864-023-09871-8

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Keywords

BMC Genomics

ISSN: 1471-2164

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