Odorant binding proteins (OBPs) with high antennal expression levels have been widely identified among insect species since 1981 (Vogt and Riddiford, 1981). The widespread distribution of these proteins, together with their broad affinities for plant volatiles as well as pheromones in vivo, suggest critical roles in peripheral signal transmission for foreign olfactory ligands. Compared with other insects, aphids generally express fewer OBPs (between nine and twelve), such as Acyrthosiphon pisum with 11 identified genes encoding complete OBPs (Zhou et al., 2010; Biasio et al., 2015), Aphis gossypii with nine (Gu et al., 2013), Myzus persicae with 11 (Ji et al., 2016), Sitobion avenae with twelve (Xue et al., 2016), Megoura viciae with 10 (Daniele et al., 2018), and Aphis glycines with 11 (Wang et al., 2021). However, at least three OBPs, namely, OBP3 (Qiao et al., 2009), OBP7 (Sun et al., 2012a) and OBP9 (Qin et al., 2020), exhibit properties as E-β-farnesene (EBF)-binding proteins in aphids. The release of alarm pheromone signals enables aphids to escape and defend themselves (Nault et al., 1973; Pickett and Griffiths, 1980). The high proportion of EBF-binding proteins (three out of the nine to twelve OBPs) indicates the importance of alarm pheromones in aphid survival and population expansion.

Spatial expression profiles showed that insect OBPs tended to be expressed in chemosensory organs such as antennae, heads and legs (e.g., Vogt and Riddiford, 1981; Sun et al., 2013). Besides, they occur in non-sensory tissues and organs such as the wings (Calvello et al., 2003; Pelosi et al., 2005), reproductive organs (Li et al., 2008; Sun et al., 2012a), mandibular glands (Iovinella et al., 2011) and salivary glands (Zhang et al., 2017) as well. Work on OBPs in aphids has also been widely published, including studies focusing on expression analysis between winged and wingless morphs (e.g., Gu et al., 2013; Xue et al., 2016; Wang et al., 2019, 2021). For example, Wang et al. (2021) conducted a systematic and detailed investigation of OBP expression in the antennae, head, wings, legs, cornicles, caudae, and thorax of the soybean aphid A. glycines through differential transcriptome analysis and quantitative real-time PCR (RT-qPCR).

The temporal expression profile of aphids has mainly been compared between the nymph and adult stages (e.g., Xue et al., 2016; Wang et al., 2019). The expression profiles revealed higher levels of some OBPs in the nymph stage than in adults. However, considering that the body undergoes a substantial transformation from the nymph to adult stage and that the adult stage lasts up to an average of 19 d (Li et al., 2018), a detailed investigation of key time points such as the beginning of the adult stage and several time points during the whole adult stage will hopefully clarify the temporal expression patterns of OBPs throughout the adult stage. To date, there has been a lack of research on this topic.

Wing state is a phenotypic correlate of olfaction sensitivity in aphids, with the winged morph displaying greater sensitivity (Pickett, 2009). Winged aphids play more important roles in risk avoidance, migration, and habitat reselection over long distances, whereas wingless aphids are responsible for rapid population expansion after colonization. Therefore, additional investigations of the expression pattern of OBPs between the two wing phenotypes will refine the study of temporal expression patterns. In addition, such investigations would help reveal the potential function of OBPs from a novel perspective.

Insect hyperawareness of potential food, mates or danger is mostly due to the highly sensitive insect chemosensory system. Intense responses of OBPs to chemical stimuli have been widely reported (e.g., Pelosi et al., 2005; Siciliano et al., 2014), but how intense the response of OBPs to newly occurring odors is in aphids remains an open question. Before answering this question, we must answer a more fundamental one: under constant conditions, what are the temporal expression patterns of OBPs during the aphid adult stage?

In the present study, we carried out a detailed investigation of the temporal expression profile of EBF-binding proteins in the grain aphid Sitobion miscanthi, the most widespread and harmful pest and dominant aphid species of wheat in China, at the beginning, middle and end of the adult stage. In China, S. miscanthi was wrong using for the Latin name as S. avenae (Zhang, 1999; Jiang et al., 2019). The time points employed in the survey were 0, 2 h, 1 day, 3 days, 10 days, and 20 days after the emergence of adults. Furthermore, we collected antennae from both winged and wingless individuals to determine whether wing morph is a phenotypic correlate of OBP expression. Finally, the expression of these genes before and after EBF induction was analyzed by carrying out an EBF treatment experiment.

OBP3 in the pea aphid A. pisum was the first reported EBF-binding protein as well as the first OBP reported in aphids (Qiao et al., 2009). Several studies have since reported that OBP7 also shows a specific affinity for EBF in the pea aphid A. pisum and peach aphid M. persicae (Sun et al., 2012b), grain aphid S. avenae (Zhong et al., 2012) and bird cherry oat aphid Rhopalosiphum padi (Fan et al., 2017). More recently, OBP9 was reported to have a broad affinity spectrum including EBF (Qin et al., 2020). To date, at least three EBF-binding proteins have been reported, which suggests that peripheral transmission of EBF may be achieved through the interaction of multiple OBPs.

In the present study, in early adulthood, we found two trends of OBP expression, namely, stable expression (OBP3 in the wingless morph, OBP9 in the winged and wingless morphs, and OBP10 in the wingless morph, Figure 1) and upregulated expression (OBP3 and OBP10 in the winged morph and OBP6 and OBP7 in the winged and wingless morphs, Figure 1), and there was no downregulated expression. By late aphid adulthood, even including the very end of the life cycle (20 d), the expression levels of the five OBPs all remained steady. Our investigation revealed complex expression level patterns for different OBPs during the aphid adult stage. Therefore, to clarify the expression patterns of aphid OBPs, we suggest subdividing the adult stage into different periods and including 0 h as a sampling time point. An additional odorant, i.e., EBF, was able to further stimulate the expression of corresponding OBPs, which obviously increased the complexity of the expression patterns.

Interestingly, among the three EBF-binding proteins, only OBP7 was significantly more highly expressed in the antennae of winged aphids than in wingless aphids. Within the investigated temporal range, the antennal expression levels of this protein were regularly dynamic but remained higher in the winged morph than in the wingless morph at all time points. However, there was a lower but continuously stable expression level in the wingless morph. However, OBP3 was found to have a novel expression pattern between the wing morphs. There was a significantly lower level of antennal OBP3 expression in newly emerged winged aphids (at the P = 0.05 level), which increased rapidly to the same level as that in the wingless aphids at 1 day and remained steady and equivalent to that level over time. This is in sharp contrast with the patterns observed for OBP6, OBP7, and OBP10 (sustained higher expression level in the winged morph, Figure 1) and OBP9 (sustained high expression level in both wing morphs with no significant difference, Figure 1). Similar to that of OBP3, the expression of OBP9 showed no phenotypic difference. OBP9 was the only OBP that did not exhibit an expression trough at the beginning of the adult stage (0 h). This OBP showed relatively stable and high expression throughout the adult stage and the highest expression level among the three EBF-binding proteins (Figure 2). After EBF induction, the expression of this OBP was further upregulated in both morphs (Figures 3, 4). The above results indicated that this protein may be an important molecule for EBF recognition in aphids among two wing morphs. Antennal expression of OBP7 strongly responded to EBF but only in the winged morph (Figures 3, 4), suggesting that this protein plays an important role in the more sensitive EBF recognition process of winged aphids. In addition, the expression level of OBP3 in antennae did not respond to EBF induction. It remained at a low level in the antennae of both the winged and wingless phenotypes (Figures 1–4). According to previous work showing that OBP3 is a systemic OBP with stronger expression in multiple tissues and organs, such as the cornicles and caudae (Xue et al., 2016; Wang et al., 2021), we speculated that OBP3 may play a limited role in the olfactory recognition of EBF and may play a carrier role for EBF in EBF storage organs, such as cornicles. The phenotypic specificity of OBP expression has been reported in different studies with various results, showing highly complex patterns that are difficult to parse. For example, the expression level of OBP7 in the winged morph did not increase to a relatively high level until 1 day. Assuming the sampling time is 0 or 2 h or a mixture of samples is sampled at various time points, the results may vary or even seem contradictory. Furthermore, the antennal expression level of OBP7 in the winged aphids at 0 or 2 h (before the peak at 1 day) was the same as that in the wingless aphids during the whole adult stage after 1 day. Thus, once samples at these two time points were selected, it was not surprising that OBP7 expression was not significantly different between winged and wingless aphids, as previously reported in some studies (e.g., Xue et al., 2016; Wang et al., 2019). As after emergence, most OBP genes undergo a process of increased expression and then stabilized, and the time to stabilize was about 1–3 days after emergence (Figure 1). Our analysis lead to the conclusion that treating the whole adult stage as a single stage in studies on expression levels does not provide sufficient resolution. In general, the repeatable and reliable expression level can be obtained 1–3 days after adult emergence when the OBP expression level is stable.

Similar to that of OBP7, the expression of OBP6 peaked at 1 day in both morphs and then remained steady in the later adult stage, and OBP10 presented expression patterns similar to that of OBP7 as well (Figures 1, 2).

In summary, our findings will be helpful for understanding the interaction mode of the three EBF-binding proteins mediating EBF perception in aphids. Obviously, both phenotypes possess a molecular basis (OBP9) for ordinary EBF perception by the antennae. Furthermore, OBP7 may enable greater sensitivity to EBF in winged aphids because of its significantly higher expression level in these aphids than in wingless aphids. Further functional studies are needed to clarify why OBP3, which has an EBF affinity, does not respond to EBF induction.

The above findings revealed the detailed temporal expression patterns of OBPs in aphids. They showed that figuring out the temporal expression patterns is critical for correctly selecting morphs and sampling times and will help researchers obtain reliable findings and draw solid conclusions.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

JF conceived and designed the study. JF and JC supervised the project. SZ and QZ carried out the laboratory work. SZ, JF, XJ, YQ, and WW performed the RT-qPCR data analysis. SZ, JF, XJ, and QL worked on the statistical analysis and charting. JF and SZ wrote the original manuscript. All authors contributed on polishing and revising the work.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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