Odorant-Binding Proteins and Chemosensory Proteins in Spodoptera frugiperda: From Genome-Wide Identification and Developmental Stage-Related Expression Analysis to the Perception of Host Plant Odors, Sex Pheromones, and Insecticides

Abstract

Spodoptera frugiperda is a worldwide generalist pest with remarkable adaptations to environments and stresses, including developmental stage-related behavioral and physiological adaptations, such as diverse feeding preferences, mate seeking, and pesticide resistance. Insects’ odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are essential for the chemical recognition during behavioral responses or other physiological processes. The genome-wide identification and the gene expression patterns of all these identified OBPs and CSPs across developmental stage-related S. frugiperda have not been reported. Here, we screened for genome-wide SfruOBPs and SfruCSPs, and analyzed the gene expression patterns of SfruOBPs and SfruCSPs repertoires across all developmental stages and sexes. We found 33 OBPs and 22 CSPs in the S. frugiperda genome. The majority of the SfruOBP genes were most highly expressed in the adult male or female stages, while more SfruCSP genes were highly expressed in the larval or egg stages, indicating their function complementation. The gene expression patterns of SfruOBPs and SfruCSPs revealed strong correlations with their respective phylogenic trees, indicating a correlation between function and evolution. In addition, we analyzed the chemical-competitive binding of a widely expressed protein, SfruOBP31, to host plant odorants, sex pheromones, and insecticides. Further ligands binding assay revealed a broad functional related binding spectrum of SfruOBP31 to host plant odorants, sex pheromones, and insecticides, suggesting its potential function in food, mate seeking, and pesticide resistance. These results provide guidance for future research on the development of behavioral regulators of S. frugiperda or other environmentally friendly pest-control strategies.

1. Introduction

Insects are among the organisms with robust behavioral and physiological adaptations to complex and competitive ecosystems [1,2]. Among their various sensory capacities, insects use chemosensory perception to initiate several behavioral and physiological responses [3]. For sensing chemicals, several chemosensory neurons house specific sensilla that are located on different body parts of the insect, including the antennae, wings, mouthparts, ovipositor, legs, and other appendages [4]. In general, odors and tastant molecules diffuse through epidermal pores on the sensillar surface, and they bind to odorant-binding proteins (OBPs) or to chemosensory proteins (CSPs), also known as olfactory segment D-like proteins (OS-Ds), which are soluble proteins that are secreted at high concentrations in the sensillar lymph and are responsible for bringing odors and tastants to specific chemosensory receptors [5].

The insect’s antenna is one of the main organs on which the expression of binding proteins, like OBPs and CSPs, play important functions in chemosensation and, as a consequence, in the behavioral modulation of the insect [6]. In this and other chemosensory organs, the role of OBPs and CSPs is to deliver ligands to different chemosensory receptors, including olfactory receptors (ORs), ionotropic receptors (IRs), gustatory receptors (GRs), or sensory neuron membrane proteins (SNMPs), which are situated in the dendritic membrane of the olfactory neurons [7,8]. These, in turn, are associated with the opening of cation channels for converting the chemical signal to an electrochemical signal, which is the basis of the neuronal modulation for behavioral and other physiological responses [9]. After the activation of the chemosensory receptors, odorant degrading enzymes (ODEs) rapidly degrade the odorants [10]. However, other studies have demonstrated that the expression of odorant-binding proteins in other tissues such as the salivary glands, fat body, or midgut may be associated with alternative physiological functions in the insect [11,12,13]. Some OBPs, for instance, have been found to relate to innate immunity, pesticide resistance, and anti-inflammation [14,15,16]. The expression of CSPs has been reported in diverse insect tissues [17,18] to function in pheromone delivery, nutrient transportation, visual pigmentation, development, immunity, or pesticide resistance [19,20,21,22,23].

Both OBPs and CSPs are water-soluble, low-molecular-weight acidic proteins of approximately 13–17 kDa with a polypeptide length of about 120–170 amino acid residues [24,25]. OBPs are provided with an asset of conserved cysteine (Cys) residues [26] distributed within a hydrophobic cavity formed by at least six α-helix domains [27], which represents a conserved structural feature among the OBPs of most insects. In general, based on the number of conserved Cys, insect OBPs can be divided into 5 types: (1) classical OBPs with 6 conserved Cys, (2) plus-C OBPs with 8 conserved Cys, (3) minus-C OBPs with 4 conserved Cys, (4) dimer OBPs with 12 conserved Cys, and (5) atypical OBPs with 9–10 conserved Cys [28]. OBPs in general follow a regular pattern alternating cysteines with other amino acids (C-pattern), which is more or less conserved to facilitate the binding to ligands [29,30]. Varying among different orders, the following pattern, C1-X25-30-C2-X3-C3-X36-42-C4-X8-14-C5-X8-C6, where X is any amino acid, has been described in Lepidoptera, and a few cases have been recently reported in Spodoptera exempta [31].

Most insect CSPs, instead, have four conserved cysteine residues forming two disulfide bridges [32]. In lepidopterans, the C-pattern of CSPs is C1-X6-C2-X18-C3-X2-C4 [33]. Compared with OBPs, the conservation of CSPs is more distinct: insect CSPs maintain a high homology within species, between different orders, and across families. For example, CSPs are 50% to 60% homologous between Schistocerca gregaria and Locusta migratoria, and 37% to 50% homologous between Orthoptera and Lepidoptera [34]. In contrast, most insect OBP sequences differ from order to order and are typically less than 20% identical between genera [35]. The expression of insect CSPs can be within (antennae) or outside the olfactory organs, with both chemosensory (e.g., taste) and nonchemosensory roles (e.g., development) [36].

Among insects, S. frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae) is a pervasive noctuid pest in the Americas and has become invasive in Africa and Asia with a life span around 30 days at 28 °C [37]. S. frugiperda has many behavioral adaptations including a broad feeding range of more than 180 plant species, a strong migratory ability, a high reproductive capacity, and a strong pesticide resistance, resulting in its being a difficult target for pest-management strategies [38,39]. Interestingly, many ways of adaptation of this insect are dependent on its developmental stage. For instance, young larvae of S. frugiperda prefer to feed on cotton leaves, and they progressively change their preferences to fruiting structures, like squares and bolls, in older instars [40]. Older S. frugiperda larvae display cannibalism and stronger microbial and chemical pesticide resistance than younger larvae [41,42]. Male mate-seeking and female egg-laying site selection behaviors are common behavioral adaptations in these species by metamorphosis into adults with flying ability [43]. As a consequence, the various developmental and sex-related behavioral and physiological adaptations of this species have enabled its fast global spread and severe damage to various crops. Given the broad evidence of the diverse behavioral and physiological functions of OBPs and CSPs in insects, a genome-wide investigation and a better understanding of the phylogenetic relationships, gene structure, and the expression patterns of these proteins in S. frugiperda is essential.

Recently, the in-sight/in-depth view of the genome of S. frugiperda has been released [44], but the chemosensory proteins have previously been identified only based on transcriptomic data [45,46]. By providing a wide genomic investigation, we identified an asset of S. frugiperda OBP and CSP genes to investigate their expression patterns across developmental stages and sex classes, including eggs, first to sixth instars (=larvae, in a strict sense), pupae, adult females, and males. Among the OBPs identified in this study, we reported SfruOBP31, renowned as an ortholog of D. melanogaster DmelOBP69a [47], being expressed in larvae and adults and binding to specific host plant volatiles, sex pheromones, and pesticides.

2. Results

2.1. Identification of OBP and CSP Genes in S. frugiperda

We identified 33 OBP and 22 CSP genes in the S. frugiperda sequence (

Table 1. Physical and molecular properties of 33 SfruOBPs identified in the Fall armyworm (S. frugiperda). ND: not detected.

Gene Name	Genome ID	Chromosome No.	Length (bp)	Introns	Exons	Amino Acids (aa)	Molecular Weight (kDa)	Isoelectric Point	Signal Peptide (aa)
OBP1	Sfru136920	chr15	9606	2	3	131	14.90	9.01	ND
OBP2	Sfru112680	chr8	1607	2	3	162	18.23	5.31	1-21
OBP3	Sfru112690	chr8	1488	2	3	173	19.14	5.16	1-30
OBP4	Sfru112730	chr8	722	2	3	164	18.75	5.47	1-23
OBP5	Sfru220490	chr10	10,632	2	3	146	16.16	4.47	1-21
OBP6	Sfru220520	chr10	1035	2	3	147	15.73	5.42	1-21
OBP7	Sfru220480	chr10	2148	2	3	121	13.97	8.15	ND
OBP8	Sfru131490	chr2	2834	4	5	149	17.22	4.63	1-26
OBP9	Sfru078880	chr12	1052	2	3	155	18.22	4.88	ND
OBP10	Sfru072390	chr20	1203	3	4	118	12.69	8.72	1-20
OBP11	Sfru220500	chr10	5252	5	6	211	23.91	5.52	1-23
OBP12	Sfru112710	chr8	783	2	3	164	18.74	5.36	1-19
OBP13	Sfru112700	chr8	1121	2	3	209	23.92	8.07	1-22
OBP14	Sfru220470	chr10	12,205	5	6	191	21.37	5.84	ND
OBP15	Sfru112570	chr8	644	2	3	164	19.27	5.51	1-19
OBP16	Sfru136280	chr15	852	1	2	159	18.58	8.46	1-19
OBP17	Sfru036850	chr22	3719	4	5	141	15.70	6.86	ND
OBP18	Sfru153150	chr22	5083	4	5	135	14.44	8.96	ND
OBP19	Sfru036860	chr22	2766	3	4	202	22.75	5.12	1-19
OBP20	Sfru194150	chr15	2659	2	3	160	18.14	9.97	ND
OBP21	Sfru220550	chr10	2544	4	5	154	16.98	4.89	1-23
OBP22	Sfru221430	chr25	1817	3	4	156	16.93	7.51	1-22
OBP23	Sfru131450	chr2	14,905	5	6	241	27.63	5.82	ND
OBP24	Sfru032040	chr3	14,476	4	5	156	17.81	6.38	ND
OBP25	Sfru220530	chr10	1592	3	4	152	16.32	4.69	1-21
OBP26	Sfru220540	chr10	635	1	2	148	16.15	4.89	ND
OBP27	Sfru116380	ctg319_4	635	0	1	211	24.78	6.91	ND
OBP28	Sfru007560	chr8	9327	2	3	244	26.94	5.61	1-18
OBP29	Sfru220560	chr10	6574	7	8	272	30.59	5.16	1-20
OBP30	Sfru136290	chr15	3781	2	3	136	15.53	7.63	1-19
OBP31	Sfru155250	chr14	413	0	1	137	14.71	4.36	1-20
OBP32	Sfru139020	chr25	2713	3	4	133	15.09	8.88	1-16
OBP33	Sfru220510	chr10	1769	4	5	153	17.10	4.72	1-21

and

Table 2. Physical and molecular properties of 22 SfruCSPs identified in the Fall armyworm (S. frugiperda). ND: not detected.

Gene Name	Genome ID	Chromosome No.	Length (bp)	Introns	Exons	Amino Acids (aa)	Molecular Weight (kDa)	Isoelectric Point	Signal Peptide (aa)
CSP1	Sfru089520	chr8	18,161	4	5	233	25.59	10.44	ND
CSP2	Sfru158600	chr19	3201	2	3	107	11.94	9.49	1-22
CSP3	Sfru069670	chr8	9467	1	2	127	14.80	6.74	1-18
CSP4	Sfru007770	chr8	5864	1	2	123	13.57	5.23	1-18
CSP5	Sfru069550	chr8	3841	1	2	131	15.61	9.42	1-25
CSP6	Sfru007800	chr8	3306	1	2	114	12.92	5.04	1-16
CSP7	Sfru069640	chr8	2542	1	2	128	14.60	5.43	1-16
CSP8	Sfru069600	chr8	2859	1	2	120	13.77	5.86	1-16
CSP9	Sfru069650	chr8	2240	1	2	123	14.36	6.82	1-18
CSP10	Sfru069590	chr8	2891	2	3	166	18.59	9.57	ND
CSP11	Sfru007780	chr8	1057	1	2	122	13.82	8.95	1-16
CSP12	Sfru007430	chr8	2617	1	2	126	14.10	7.65	1-15
CSP13	Sfru007750	chr8	853	1	2	123	13.78	8.77	1-16
CSP14	Sfru069660	chr8	1679	1	2	122	14.20	6.12	1-16
CSP15	Sfru069630	chr8	3308	1	2	156	18.30	9.04	ND
CSP16	Sfru007790	chr8	2358	2	3	151	17.14	5.61	ND
CSP17	Sfru069620	chr8	828	1	2	122	14.03	5.84	1-17
CSP18	Sfru069680	chr8	6950	2	3	203	23.31	6.90	ND
CSP19	Sfru158610	chr19	3462	2	3	116	12.87	9.38	1-29
CSP20	Sfru069530	chr8	735	1	2	122	13.62	5.14	1-17
CSP21	Sfru069700	chr8	1054	1	2	153	17.12	8.86	ND
CSP22	Sfru069690	chr8	1857	3	4	170	19.22	4.93	ND

). The OBP sequence multiple alignment performed with DNAMAN was 12.43%, while the CSP was 24.91%. All the OBPs we have found have conserved cysteines C3 and C4, except for OBP18 and OBP23. Twenty-five OBPs belong to the classical OBP group (SfruOBP1–14, 16, 19–21, 24, 26–28, 30, 31, and 33), provided with six conserved cysteine residues. Among them, SfruOBP10, 16, 19, 20, 27, 28, and 30 miss the conserved C1, and SfruOBP9 misses the conserved C2, although classified as a classical OBP given the conservation of the other five conserved cysteines. Three of the OBPs we have identified belong to the minus-C OBP group (SfruOBP17, 22, and 32), provided with four conserved cysteines, missing C2 and C5. SfruOBP15, 25, and 29 have six conserved cysteines, and they also carry an extra cysteine downstream of C6, being classified as Plus-C OBPs. For SfruOBP18, provided with only the conserved C5, and for SfruOBP23, missing conserved cysteines, we cannot determine which OBP group they belong to, although we have identified OBP genes with a high homology with them in NCBI (Supplemental Table S1). Contrary to SfruOBPs, SfruCSPs are highly conserved, provided with four conserved cysteines except for SfruCSP1, which had no conserved cysteines; however, blast in NCBI revealed a CSP with a high homology, so it was identified as a putative CSP (Supplemental Table S1).

Gene lengths of SfruOBPs ranged from 413 bp (SfruOBP31) to 14,905 bp (SfruOBP23), with an average length of 3897 bp. The length of the CSP genes ranged from 735 bp (SfruCSP20) to 18,161 bp (SfruCSP1), with an average length of 3688 bp. The protein sequences encoded by the OBP genes ranged from 118 (SfruOBP10) to 272 (SfruOBP29) amino acids, with an average length of 167 amino acids. The protein sequences encoded by the CSP genes ranged from 107 (SfruCSP2) to 233 (SfruCSP1) amino acids, with an average length of 139 amino acids. The molecular weight of the SfruOBP proteins ranged from 12.69 kDa (SfruOBP10) to 30.59 kDa (SfruOBP29). The theoretical isoelectric points ranged from 4.36 (SfruOBP31) to 9.97 (SfruOBP20), respectively. The molecular weight of the SfruCSPs proteins ranged from 11.94 kDa (SfruCSP2) to 25.59 kDa (SfruCSP1). The theoretical isoelectric points ranged from 4.93 (SfruCSP22) to 10.44 (SfruCSP1). Most of the OBPs/CSPs (SfruOBP2-6/8/10-13/15/16/19/21/22/25/28-33, SfruCSP2-9/11-14/17/19/20) had a signal peptide ranging from 15 to 30 amino acids at their N-terminal, as evidence of their classification among the secretory proteins.

2.2. Phylogenetic Analysis of OBP and CSP in S. frugiperda

Phylogenetic analysis of the OBPs (Figure 1) demonstrated the existence of five clusters based on the homology among the representatives. Cluster 1 and Cluster 5 contain most of the SfruOBPs. Cluster 3 is the second largest cluster, containing five SfruOBPs, while both Clusters 2 and 4 contain three SfruOBPs. Most SfruOBPs are in the same subclade with SlitOBPs (Supplemental Table S2), as an indication of the close evolutionary relationship between the two species.

Phylogenetic analysis of the CSPs (Figure 2) demonstrated the existence of four clusters based on the homology among the different representatives. Clusters 2 and 4 contain most of the SfruCSPs (12 + 7); among them, most CSPs of Cluster 2 belong to lepidopterans. Interestingly, Cluster 3 contains only CSPs from S. invicta (SinvCSP1-5/8/10/13-15/17-20). Cluster 1 contains the remaining three SfruCSPs (SfruCSP1/8/21) and four LmigCSPs (LmigCSP10/26/27/31). Like SfruOBPs, most SfruCSPs that are in the same subclade with SlitCSPs show a close evolutionary relationship to S. litura CSPs (Supplemental Table S3), followed by the CSPs of B. mori.

2.3. Chromosomal Distribution of OBP and CSP Genes

The chromosomal location map of OBP and CSP genes was plotted with tbtools based on the information of the genome annotation file of S. frugiperda (Figure 3). The identified OBP genes of S. frugiperda are located on 10 different chromosomes and on contig ctg319_4 (which is a different contig not yet defined, belonging to a specific chromosome), and the majority of the SfruOBPs are distributed in clusters on chromosomes. For instance, nine genes from Cluster 1 (SfruOBP5/6/11/14/21/25/26/29/33) are located in proximity on chromosome 10, while the remaining two genes of Cluster 1 are instead distributed on chromosome 20 (SfruOBP10) and chromosome 22 (SfruOBP19). One SfruOBP gene from Cluster 2 is located on chromosome 2 (SfruOBP23), and two SfruOBP genes from the same Cluster are located on chromosome 22 (SfruOBP17/18). The SfruOBP genes from Cluster 3 are distributed dispersedly on chromosome 2 (SfruOBP8), chromosome 10 (SfruOBP7), chromosome 14 (SfruOBP31), and chromosome 25 (SfruOBP22/32), respectively. Two SfruOBPs (SfruOBP1/20) from Cluster 4 are located on chromosome 15 and another one (SfruOBP27) is located on ctg319_4. Six SfruOBPs from Cluster 5 (SfruOBP2/3/4/12/13/15) are located on the adjacent segments of chromosome 8 as well as another OBP gene (SfruOBP28), which is located on the same chromosome but at approximately 3 Mbp distance from the latter. Other OBP genes of Cluster 5 (SfruOBP16/30) are located on chromosome 3 (SfruOBP24), chromosome 12 (SfruOBP9), and chromosome 15 (SfruOBP16/30).

Contrary to SfruOBPs, the distribution of SfruCSPs seems to be more centralized, having identified loci on only two chromosomes: two genes of Cluster 2 (SfruCSP2/20) are located on adjacent loci of chromosome 19, while the remaining 21 genes of Clusters 1, 2, 3, and 4 are all located on chromosome 8 (Figure 4). On this chromosome, most of the genes fall in proximal loci, with the exception of SfruCSP1 and SfruCSP12, located at 3.5 Mbp and 6.6 Mbp, respectively, on the chromosome.

2.4. Intron/Exon Organization of OBP and CSP Genes of S. frugiperda

The exon/intron structure varies greatly within SfruOBP (Figure 5) and SfruCSP (Figure 6) genes from different clusters. For example, SfruOBP2/3/4/9//12/13/28/30 from Cluster 5 have three exons each, while SfruOBP24 has five exons. SfruOBP29 (Cluster 1) contains eight exons, which is the highest number of exons. However, two genes contain only one big exon (SfruOBP27/31). Members of the SfruCSPs have fewer exons than those of the SfruOBPs. The majority of the SfruCSP genes have only two exons, except for SfruCSP2/10/16/18/20 with three exons, SfruCSP22 with four exons, and SfruCSP1 with five exons. When compared, the exon and gene lengths of the SfruCSP members differed from those of the SfruOBP members.

2.5. Motif Analysis of OBPs and CSPs of S. frugiperda

MEME analysis revealed that there are six different motif structures in the polypeptide sequences of SfruOBPs (Figure 7) and five different motif structures in the polypeptide sequences of SfruCSPs (Figure 8). Half of the OBPs from Cluster 1 present the 6-1-2 motif pattern (SfruOBP5/6/25/26/33), and the other half present a different organization of the motif pattern (SfruOBP11/14/19/21/29). The three SfruOBPs from Cluster 2 present only motif 1 (SfruOBP17/18/23) as well as nine other SfruOBPs (SfruOBP1/7/9/10/19/20/23/24/28). Three SfruOBPs from Cluster 3 present the 6-1 motif pattern (SfruOBP8/31/32) that is present also for SfruOBP27 from Cluster 4, while SfruOBP22 presents the 1-6 motif pattern and SfruOBP7 presents only one motif (motif1). OBPs from Cluster 5 (SfruOBP2/3/4/12/13/15) present the 4-1-5-3 motif pattern.

The majority of the SfruCSPs present a pattern of 4-3-1-2 motifs (Figure 8). These included SfruCSP8 from Cluster 1, almost of the SfruCSPs from Cluster 2, as well as all the SfruCSPs from Cluster 4. The remaining five SfruCSPs showed four distinct motif patterns (SfruCSP1/2/6/20/21). Among the latter, two SfruCSPs from Cluster 2 present the 4-1-5 motif pattern (SfruCSP2/20), SfruCSP6 presents only motif 4, SfruCSP21 presents the 4-1 motif pattern, and SfruCSP1 presents only motif 2.

2.6. Gene Expression Analysis of OBPs and CSPs of S. frugiperda

Our transcriptomic dataset was used to create a heatmap revealing expression levels of SfruOBPs and SfruCSPs across the developmental stages of egg, first to sixth instars (identified by the size of the larvae), and pupae, as well as adult males and females (Figure 9). Transcriptomic analysis revealed that the expression of almost all of the genes of Cluster 1 was higher in the first instar and decreased in the second to sixth instars and the pupal stage, but recovered to a higher level in the adult stage. However, for SfruOBP19, expression appeared stable in both the larval and adult stages, and SfruOBP11 showed high expression only in the first instar and maintained a low expression for all the other developmental stages. Two genes in Cluster 2 (SfruOBP17/18) were stably expressed at all stages except eggs and the first instar, while SfruOBP23 was expressed at instar one through five and showed a decreasing trend and absent to low expression from the pupal to adult stages. The expression of SfruOBP7 and SfruOBP31 from Cluster 3 was higher in the first instar. Three other genes (SfruOBP8/22/32) were highly expressed in the pupae and adults. Two genes of Cluster 4 (SfruOBP20/27) and two genes of Cluster 5 (SfruOBP16/30) were highly expressed in both the male and female adults. SfruOBP20 was also highly expressed in the first and fourth instars, and SfruOBP1 in Cluster4 was highly expressed in the first to third instars. Some of the genes from Cluster 5 were highly expressed in the egg stage, while all genes from this cluster were highly expressed in adults. Genes of Cluster 5 were also moderately to highly expressed in several earlier or later larval stages: SfruOBP3/13/15 were highly expressed in earlier larval stages (instars 1–3), SfruOBP4/12 were highly expressed in later larval stages (instars 4–5), while SfruOBP2 was expressed in all instars, and SfruOBP9/24/28 were also expressed in most stages of the larva. It is worth mentioning that SfruOBP9 was highly expressed in males but low in females.

The majority of the SfruCSPs showed a more complex expression tendency across the developmental stages and sexes (Figure 10) when compared with SfruOBPs. In Cluster 1, SfruCSP21 gradually decreased in expression level from eggs to the sixth instar but was highly expressed in pupae and adults, SfruCSP8 was highly expressed only in adults, and SfruCSP1, from a low to absent expression in eggs, maintained a moderate expression in all developmental stages. All the genes from Cluster 2 were moderately or highly expressed from the egg to pupal stages. However, SfruCSP11/16 showed low expression in the sixth instar, SfruCSP17 showed low expression in the fourth to sixth instars and in the egg stage, SfruCSP5 showed low expression in the fourth instar, and SfruCSP4 showed a moderate expression for all larval stages except for the sixth instar. Two genes showed a relatively low expression in pupae (SfruCSP6/13), and only three genes of this cluster exhibited high expression levels in adult males and females (SfruCSP4/6/17). Within the same cluster, SfruCSP2, after its high expression in eggs, gradually increased expression levels from the first instar to the pupal stage, dropping to low levels in adults. Within the same cluster, SfruCSP6 showed an overall high expression except for the first instar and the pupae, while SfruCSP20 showed a sort of off-on expression pattern throughout all the developmental stages of the insect, from eggs to adults. Except for SfruCSP15/22, all genes from Cluster 4 were highly expressed from the first instar to the fifth instar, where SfruCSP18 showed a reduced expression. Expression of the genes from Cluster 4 seems to be consistent in both adult males and females. Interestingly, there was low expression of SfruCSP7/9/14 in pupae and of Sfru3/7/18 in the sixth instar. SfruCSP22 was highly expressed in adult females and showed a lower expression across the other developmental stages.

Among the examined genes, the expression patterns of SfruOBP32 (Figure S1A), SfruCSP3 (Figure S1B), SfruCSP19 (Figure S1B), and SfruCSP20 (Figure S1B) were similar across all developmental stages comparing the qRT-PCR data with the transcriptomic data. The expression of SfruOBP24 (Figure S1A) in four stages (fifth instar, sixth instar, pupae, and female adult) was consistent between the qRT-PCR and transcriptomic data, but the qRT-PCR analysis also revealed moderate expression of SfruOBP24 at the fourth instar, third instar, first instar, and egg stages and high expression of SfruOBP24 at the second instar and the male adult stages. The qRT-PCR results revealed an adult male biased expression pattern of SfruOBP22. The expression of five stages (L2, L3, L4, L6, and AM) of SfruOBP17 was also consistent between the qRT-PCR and transcriptomic data (Figure S1A).

2.7. Binding of SfruOBP31 to Host Volatiles, Pheromones, and Pesticides

SfruOBP31 showed binding capacity to 1-NPN with a Ki of 22.4 μM, indicating 1-NPN as a proper fluorescence reporter (Figure 11A). A chemical-competitive binding assay unveiled SfruOBP31 binding to linalool, cis-3-hexenyl acetate, 1-nonanol, and decanal, with the following Ki values: 4.04, 6.38, 14.17, and 19.56 μM, respectively. Among sex pheromones, SfruOBP31 binds (Z)-11-hexadecenyl acetate and (Z)-9-tetradecenyl acetate with Ki values of 5.40 and 5.02 μM, respectively, but not to (Z)-7-dodecenyl acetate or (Z)-9-dodecenyl acetate. Among insecticides, we reported SfruOBP31 binding to lambda-cyhalothrin (Ki = 2.78 μM) and chlorfenapyr (Ki = 8.1), but a lack of binding to emamectin benzoate (Ki = 27.55 μM), lufenuron (Ki = 37.58 μM), and chlorantraniliprole (Ki= 22.28 μM).

3. Discussion

S. frugiperda is an important and invasive agricultural pest whose destructiveness occurs across life stages, behavior patterns, and physiological changes [48]. In this paper, by performing a genome-wide screening of S. frugiperda, we have unveiled its asset of OBPs and CSPs. Based on a phylogenetic analysis of SfruOBPs and SfruCSPs, we compared the gene structures, polypeptide sequence motif patterns, and patterns of gene expression across all developmental stages of the insect, from eggs to adults. We chose one of the highest expressed binding proteins of S. frugiperda, SfruOBP31, that we heterologously expressed and purified from transgenic E. coli (Figure S2) to assess chemical-competitive binding assays demonstrating binding to several ligands among odors emitted from the host plant Z. mays, sex pheromones, and some of the main pesticides for this insect.

In the last decades, most of the investigation of lepidopterans targeted their chemoreceptors [49,50,51,52]. Among various reports, some provided in-depth analysis of specific OBPs [53,54], CSPs [18,55], or both [30,56]. However, to our knowledge, this is among the few [57], or may be the sole study in lepidopterans, in which a systematic investigation of soluble proteins merges with a variegate analysis including genomic loci, gene and polypeptide structures, expression patterns across every developmental stage, and functional evidence of semiochemical/pesticide binding.

By mediating the ligand transport through the sensillar endolymph to the activation of chemosensory transmembrane proteins [7,8], OBPs and CSPs play a crucial role in insect behavioral and physiological adaptation, including food seeking, reproduction, and pesticide resistance [58].

Belonging to two multigene families, OBPs and CSPs generally differ in numbers and functionalities across insect species [21]. To our knowledge, this is among the first studies identifying OBPs and CSPs with the in-sight/in-depth view of the genome of S. frugiperda, wherein we demonstrate the existence of 33 genes for OBPs and 22 genes for CSPs. Our findings are consistent with a previous study conducted on S. frugiperda where 36 OBPs and 21 CSPs were identified [45], with 22 SfruOBPs and 19 SfruCSPs identical with our study (Supplemental Table S4) and comparable with the number of the respective genes for S. exigua (34 OBPs, 20 CSPs) [59].

Taking all binding proteins together, S. frugiperda expresses fewer OBPs and CSPs than B. mori (44 OBPs, 20 CSPs) [33,57] and S. litura (45 OBPs, 23 CSPs) [60]. The SfruOBPs were classified according to Hekmat-Scafe et al. [61]. SfruOBPs with Cys at only six conserved sites were classified as classical OBP, while SfruOBPs in the absence of C2 and C5 were classified as minus-C OBP. SfruOBPs with more than six conserved Cys were classified as plus-C OBPs. Among the 33 identified SfruOBP genes, 22 belonged to the classical group; only three SfruOBPs, which lacked C2 and C5, belonged to the minus-C group (SfruOBP22/32); and three genes belonged to the plus-C group (SfruOBP15/25/29). This is comparable to S. litura, in which only classical, minus-C, and plus-C OBPs were identified [62,63]. However, one dimer OBP was annotated in Danaus plexippus [53]. The majority of the SfruCSPs contained four conserved Cys, and only three CSPs contained three conserved Cys.

Our phylogenetic analysis demonstrates evolution relationships of OBPs and CSPs between S. frugiperda and other species of Lepidoptera; specifically, between S. frugiperda and S. litura (Figure 1). According to the phylogenetic tree, we found five clusters of SfruOBPs, among which some OBPs from the same cluster have similar gene structures and expression patterns, while some OBPs from different clusters present different gene structures and expression patterns. The phylogenetic tree of CSPs was divided into four clusters, of which only three out of four contain CSPs from S. frugiperda. Among these, Cluster 2 and Cluster 4 contain most of the SfruCSPs, as a possible indication of their similarity in evolution and possibly as evidence of differences in foraging behavior or adaptation to the environment between S. frugiperda and S. litura [64].

Chromosomal location, gene structure, and protein motif analysis may help unveil potentials from gene evolution such as duplication, reversal, or skipping [62,65] to functional conservation. In general, about half of the SfruOBPs are distributed on multiple chromosomes, whereas the SfruCSPs are distributed on only two chromosomes, and most of them are clustered on adjacent loci of one chromosome, which may be due to the high conservation of the gene family in S. frugiperda. In addition, the SfruOBP genes display divergent patterns of intron/exon organization (Figure 5), which, although speculative, may widen a potential for alternative splicing, as known for a different class of receptors in lepidopterans [66,67], and motif patterns that are only partially conserved among the representatives belonging to the same clusters (Figure 7). The evolution of the OBP gene family is suggested to follow the birth-and-death model through the pseudogenization or the functional divergence of the duplicate gene during duplication [68,69]. However, the adjacent genes on chromosomes could be involved in analogous functions. For instance, 10 of 24 OBPs located in the social chromosome of S. invicta may participate in the behavioral modulation between the monogyne colony and the polygyne colony [70]. Unlike SfruOBPs, the majority of SfruCSPs from four clusters are instead distributed in proximity on the same chromosome (chromosome 8), evidence of their possible origins from gene duplication. In addition, several SfruCSP genes display similar intron/exon organization (Figure 6), and analysis of their expressed amino acid sequences demonstrates the existence of an identical motif pattern, 4-3-1-2 (Figure 8). SfruCSP2 and SfruCSP20 are located in a separate chromosome, suggesting their divergent functions. As a homolog to SfruCSP20, SlitCSP3 was revealed to function in plant defensive metabolites recognition [71]. The functions of the chromosomal location close and far SfruOBPs/SfruCSPs are worthy of further study to verify their function evolution.

Transcriptomic analysis unveiled that six genes of OBPs of the SfruOBP-Cluster 5 (SfruOBP2/3/4/12/13/15), five genes of OBPs of the SfruOBP-Cluster 3 (SfruOBP7/8/22/31/32), two genes of OBPs of the SfruOBP-Cluster 4 (SfruOBP20/27), two genes of OBPs of the SfruOBP-Cluster 5 (SfruOBP16/30), and two genes of OBPs of the SfruOBP-Cluster 1 (SfruOBP10/19) showed high expression in adults, suggesting that they have important functions in the adult stage (Figure 9). RNA interference of highly expressed OBP1 in the adult antennae of Culex quinquefasciatus led to significantly reduced electrophysiological responses to egg-laying attractants, indicating that CquiOBP1 may mediate spawning behavior [72]. Although speculative, this may suggest potential olfactory functions of the OBPs that are highly expressed in adults’ SfruOBPs and their involvement in behaviors such as spawning but also courtship or reproduction. The majority of SfruOBPs showed low expression at the sixth instar, whereas we demonstrated higher expression for representatives of Cluster 2. We assume that the lower expression of SfruOBPs at this larval stage may be associated with delayed feeding as a prepupal physiological feature from older larvae. As expected, most SfruOBPs showed low or moderate expression in the egg stage, with the sole exception of SfruOBP12, which may be associated with some sort of role in embryo development like in Galeruca daurica, where GdauOBP28 also showed an exclusively high expression in the egg stage [73]. Contrary to our expectations, the expression of several, if not the majority, of SfruOBPs was higher in the earlier instars than in the older ones. Among these, SfruOBP11 was the OBP with the highest expression in the first instar, which, hypothetically, may suggest its role in conspecific recognition or defensive mechanisms at the larval stage. Indeed, studies have shown the role of OBP in binding chemical ligands and regulating behavior, like the larval-specific OBP of S. exigua, SexiOBP13, which showed a high binding ability to the sex pheromone, Z9, and E12-14:Ac [74]. In the pea aphid Acyrthosiphon pisum, ApisOBP3 is expressed in old larvae and in apterous adults [75] to function in short-term defensive responses like feeding cessation and dropping from host plants in a phase of the behavioral response to the alarm pheromone (E)-β-farnesene [76]. In addition, OBPs from the same clusters have similar gene structures, which can help us to classify OBPs that may have similar functions for further study. For example, SfruOBP2/3/4/12/13/15 from Cluster 5 present the 4-1-5-3 motif pattern (Figure 7), and all contain loci clustered on the same chromosome, organizing three exons, as an indication of their similarity in function.

Analyzing CSP expression, we demonstrated that most of the CSPs we identified were expressed throughout the whole life cycle of S. frugiperda, especially among the various instars (Figure 10). Compared with OBPs, CSPs were more abundantly expressed at different developmental stages, which may be associated with their broader physiological functions and tissue distribution than OBPs, being involved in leg regeneration [77] and sucking [20]. The expression of some of the genes from Cluster 2 (SfruCSP2/5/10/11/12/13/16/19/20) was lower in the adult stages, but in comparison, it was higher in the later larval stages. These genes may be related to the developmental-stage-dependent behavioral or physiological adaptations in older larvae. Genes of Cluster 4 (SfruCSP15/22) and SfruCSP8 were highly expressed during adulthood, indicating a function in behaviors such as courtship, oviposition, or reproduction [78]. For example, the adult expression with a female bias of SfruCSP22 suggests this gene’s involvement in ligand binding to the neuronal modulation for behaviors forming the basis of choosing suitable sites for egg-laying or other female-specific activities [79]. Several SfruCSPs were expressed in the egg stage, among which the highest expression belonged to the CSPs from Cluster 2 (SfruCSP2/6/11/13). Studies have shown that CSP has odor recognition functions, such as the AlinCSP1-3 in the alfalfa plant bug, Adelphocoris lineolatus, binding to several host-related ligands including (Z)-3-hexen-1-ol, (E)-2-hexen-1-al, and valeraldehyde, as a possible involvement of these CSPs in host recognition [80]. Beyond chemosensation, these CSPs may have functional properties forming the basis of embryonic development, physiologically or morphologically [81]. For example, the honey bee gene AmelCSP5 is associated with the formation of the embryonic covering membrane [82].

Previous reports on Spodoptera OBPs provides a blueprint for the prediction of SlitOBP ligands based on the interaction of phylogeny and chemical structure. For example, larval SlitGOBP2 of S. litura have been reported to function in sex pheromone recognition [83]. In the same species, OBP11 displays strong binding with sex pheromones [84] and CSP8 can bind with the oviposition deterrent chemical rhodojaponin III [85]. CSP18 binds directly to chlorpyrifos/fipronil and CSP6 to chlorpyrifos, emamectin benzoate, and fipronil [86]. In addition, three CSPs of S. litura (SlitCSP11, 3, and 8) that are highly expressed in the midgut of the insect were shown to bind to host plant chemicals, and their expression levels varies depending on the host plants [80]. In this study, we verified the function of a widely expressed OBP, SfruOBP31, using a chemical-competitive binding assay based on measurements of 1-NPN-associated fluorescence (Figure 11). The reason we chose SfruOBP31 for binding assays is its high expression in the first and fifth larval instars and in adults (Figure 9). Indeed, these developmental stages were reported to be involved in some remarkable physiological and behavioral adaptions, including the dispersing behavior of the first instar [87], higher pesticide resistance in elder larvae [88], and the egg laying/mate seeking behavior of adults [89,90]. Our phylogenetic analysis clustered SfruOBP31 in Cluster 3, where, according to our transcriptomic analysis (Figure 9), it is represented by OBPs of S. frugiperda with the high expression patterns in the first instar or in the fifth instar and adults, suggesting its potential multiple functions, including the dispersing behavior in the first instar, higher pesticide resistance in elder instars, and the egg laying/mate seeking behavior of adults. Among other developmental stages, SfruOBP31 was also low to moderately expressed. In addition, SfruOBP31 is the ortholog of DmelOBP69a in Drosophila, sharing 81% query cover and 28.57% identity, and is involved in social interactions by the detection of contact sex pheromones [47]. Among lepidopterans, sex pheromones are key to mating behavior in adults, and previous studies demonstrated that the sex pheromone mixture of S. frugiperda is composed of (Z)-9-tetradecen-1-yl acetate (Z9-14:Ac), (Z)-7-dodecen-1-yl acetate (Z7-12:Ac), (Z)-9-dodecen-1-yl acetate (Z9-12:Ac), and (Z)-11-hexadecen-1-yl acetate (Z11-16:Ac) in an 81:0.5:0.5:18 ratio [91]. Here, we tested the binding ability of SfruOBP31 to these four sex pheromones. Competitive binding assays with 1-NPN demonstrated that SfruOBP31 selectively binds with two sex pheromones ((Z)-11-hexadecenyl acetate and (Z)-9-tetradecenyl acetate); this may be due to the high content of these two compounds in the sex pheromone mixture of S. frugiperda. Binding of a larval-specific OBP to odorant pheromones is not surprising in the genus Spodoptera. Indeed, a larval-specific OBP of S. exigua, SexiOBP13, shows strong binding capacity to the female sex pheromone Z9,E12–14:Ac that can induce the attraction behavior in larvae [74]. However, evidence of the expression of this OBP also in adults with a similar level between males and females (Figure 9) may suggest involvement of this binding protein to different ecological mechanisms.

The insect olfactory system adapts to the environment by detecting specific chemical volatiles [92]. S. frugiperda can locate their host plants through plant odors. Previous studies have revealed that many plant odors attract S. frugiperda, such as linalool [93]. Therefore, we measured the binding capacity of SfruOBP31 to five host plant volatiles (Figure 11). Our findings demonstrate that our host plant odorants linalool, cis-3-hexenyl acetate, 1-nonanol, and decanal associate fluorescent decrement in competitive assays conducted with 1-NPN with the following Ki values: 4.04, 6.38, 14.17, and 19.56 μM, respectively. These findings provide evidence of the ability of SfruOBP31 to recognize odors emitted from host plants while the neonate larvae are emerging from the egg mass.

OBP has been shown to bind to insecticide components and to be involved in the increment of resistance. For instance, GOBP2 is responsible for chlorpyrifos tolerance in S. litura [94]. In other insects like Nilaparvata lugens, OBP3 is involved in the resistance to nitenpyram and sulfoxaflor [15]. In our binding trials we also tested the binding capacity of SfruOBP31 to five pesticides commonly used in the control of S. frugiperda, demonstrating that SfruOBP31 had the strongest binding capacity to lambda-cyhalothrin (Ki = 2.78 μM) and chlorfenapyr (Ki = 8.1 μM). Evidence of binding to pheromones, plant odorants, and pesticides suggests multiple functions for SfruOBP31 in host plant recognition and pesticide resistance, and as demonstrated for its ortholog in Drosophila, DmelOBP69a, involved in social interaction based on binding with sex pheromones [47].

In conclusion, we performed a systematic identification of SfruOBPs and SfruCSPs based on the in-sight/in-depth view of the genome and analyzed their expression patterns across all different developmental stages and adult males and females. The different expression patterns of some SfruOBPs and SfruCSPs have developmental stage specificity or sex biases, suggesting the potential functions of these genes. These results lay the groundwork for future investigation of their in vivo functions with methods such as CRISPR/Cas9 or modified RNAi [95,96]. The in vitro chemical competitive binding assay of SfruOBP31 further confirmed the involvement of some OBPs or CSPs in multiple functions such as host plant plus pheromone binding or pesticide resistance. Our study opens investigations on behavioral and physiological adaptation mechanisms of the Fall armyworm S. frugiperda for the final target to develop alternative control strategies to interfere with the behavior or other physiological functions of this pest.

4. Materials and Methods

4.1. Gene Identification

The amino acid sequences of OBPs and CSPs from B. mori, S. invicta, D. melanogaster, L. migratoria, and S. litura were selected as templates to search against the genome of S. frugiperda. Homology searching was conducted using the tBLASTn programs with the E-value cut-off of 0.0001 to retrieve alternative protein sequences of S. frugiperda OBPs and CSPs. HMMER software (version 3.0) [97] was used in search of unique domains of the corresponding gene family in the alternative protein sequences. Genes that contain a conserved PBP_GOBP domain for the OBP genes (accession number pfam01395) and a conserved OS-D domain for the CSP genes (accession number pfam03392) were maintained. These genes were then manually verified based on the conserved Cys residue characteristics of lepidopterans’ OBP genes (C1-X_25-30-C2-X₃-C3-X_36-42-C4-X_8-14-C5-X₈-C6) and CSP genes (C1-X₆-C2-X₁₈-C3-X₂-C4) [30]. After filtering the sequences without characteristic domains, the protein sequences belonging to this gene family were kept for further analysis (Table S5).

4.2. Sequence Alignment and Phylogenetic Tree Construction

The phylogenetic tree of S. frugiperda, L. migratoria, D. melanogaster, S. litura, B. mori, and S. invicta OBP and CSP genes was constructed to reveal the evolutionary relationship of SfruOBPs and SfruCSPs among species. Amino acid motifs were identified using MEME [98]. The amino acid multiple sequence alignment (MSA) was made using the muscle method with MEGA 11.0, from which we estimated the best-fitting model of amino acid substitution [99]. Phylogenetic trees were built using MEGA 11.0, where branch lengths were optimized, and branch supports were calculated using bootstrapping with 1000 replicates of neighbor joining (NJ). To facilitate future research, the OBP and CSP genes of S. frugiperda were named based on the homology of genes from other species found using blastx in NCBI.

4.3. Chromosomal Distribution, Gene Structure, and Motif Analysis of OBPs and CSPs

Chromosomal location information of SfruOBP and SfruCSP genes was downloaded from genome data [44]. We visualized the data using TBtools [100], including the location and length of the genes on the chromosome and the visualization of the exon-intron structure of the SfruOBP and SfruCSP genes. To discover the motif pattern of SfruOBPs and SfruCSPs, 33 OBPs and 22 CSPs were submitted to the MEME (version 5.5.1) online tool (https://meme-suite.org/meme/, accessed on 9 December 2022) server and a Zero-or-One Occurrence per Sequence (zoops) distribution pattern was adopted.

4.4. Samples Preparation, RNA Extraction, and RNA-seq

S. frugiperda were reared individually in plastic boxes with fresh corn leaves. Insects were maintained at 28 ± 3 °C under 70–75% relative humidity and a photoperiod of 16 h light, 8 h dark cycles. Adults of S. frugiperda were reared in 30 cm × 40 cm mesh cages supplied with fresh 10% honey water and corn seedlings for egg laying. Eggs were collected in 24 h after they were laid. Synchronized 1st-to-6th instars were collected according to their size. Three-day pupae and unmated adults were also collected. For each replication, RNA was extracted from 300 eggs and the whole body of 200, 100, 50, 10, 5, and 5 larvae at their 1st, 2nd, 3rd, 4th, 5th, and 6th stages, respectively; 5 pupae; 5 adult females; and 5 adult males. All stages were collected with three replications.

Trizol Reagent (Invitrogen, Waltham, MA, USA) was used for total RNA extraction according to the manufacturer’s instructions. Genome DNA was extracted using Turbo DNase (Thermofifisher, Waltham, MA, USA). Gel extraction was performed, and a Nanodrop ND-1000 spectrophotometer (LabTech, Hopkinton, MA, USA) was used to assess the quality and quantity of the RNA samples. RNA libraries were constructed and sequenced as described previously [101]. RNA-seq was performed by a commercial company (Lianchuan, Hangzhou, China) with a next-generation sequencing platform (Illumina NovaseqTM 6000). Differentially expressed genes across all developmental stages were analyzed using the DESeq R package (1.10.1).

4.5. q-RT-PCR Verification of Selected SfruOBPs and SfruCSPs

Beacon Designer Software (Palo Alto, CA, USA) was used to design primers (Table S6), with RPL32 (Ribosomal Protein L32) as the reference gene. Q-RT-PCR experiments were performed in accordance with the Minimum Information Required for Publication of Quantitative Real-Time PCR guidelines. A Premix Ex TaqTM II (Tli RNaseH Plus) Kit (Takara, Shiga, Japan) was used as a reagent for qRT-PCR analysis. The reaction conditions were set as follows: 95 °C for 3 min, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. Reactions were carried out in triplicate, followed by dissociation in the iCycler iQ™ Real Time PCR Detection System (Bio-Rad, Hercules, CA, USA). All developmental stages and sexes were performed with three separate biological replicates. The data were analyzed according to 2^−ΔΔCT [ΔΔCt = ΔCt (test) − ΔCt (calibrator)]. Four SfruOBPs (32/24/22/17) and three SfruCSPs (3/19/20) were selected for q-RT-PCR assay as representatives that are specifically expressed at different development stages.

4.6. Heterologous Expression and Purification of SfruOBP31 in Bacteria

We expressed SfruOBP31 from S. frugiperda in bacteria using a prokaryotic expression system. The predicted signal peptide was removed to produce a properly folded protein. The ORF (Open Reading Frame) of SfruOBP31 was cloned into a pET28a vector (Novagen, Darmstadt, Germany) and transformed into an Escherichia coli BL21 strain. Individual positive colonies were incubated until the OD (600 nm) reached 0.6-0.8. Protein expression was induced with 0.5 mM IPTG (isopropyl-β-D-1-thiogalactopyranoside) at 16 °C with overnight shaking at 220 rpm/min. Cells were harvested using centrifugation at 3000× g and resuspended in HEPES buffer (10 mM HEPES, 100 mM NaCl, pH 7.5). The supernatant and pellet were then separated after sonication and centrifugation at 14,000× g and 4 °C for 30 min. SDS-PAGE was used to demonstrate that the recombinant protein was soluble. The solution was next applied to his-trap affinity columns (Cobalt Chelating Resin, GBiosciences, St. Louis, MO, USA). Bound protein was eluted with HEPES buffer containing progressively increasing amounts of imidazole from 50 mM to 500 mM. The fractions with the recombinant protein were pooled and dialyzed three times against 3 L of HEPES buffer at 4 °C overnight after electrophoretic analysis. To confirm the size of the recombinant protein, his-tag antibody (Thermofisher, Waltham, MA, USA) was used to perform Western blot. The recombinant protein was stored at −80 °C until use.

4.7. Chemical Competitive Binding Assays

To investigate the ligand binding ability of SfruOBP31, we used a fluorescence competitive binding assay using the fluorescent probe N-phenyl-1-naphthylamine (1-NPN) [102]. Binding experiments were performed on a microplate reader using a Greiner 96 Black Flat Bottom. Among the ligands, we tested five host volatiles from the host plant Zea mays: decanal (97% purity, Shanghai Acmec Biochemical Co., Ltd., Shanghai, China), linalool (98% purity, Shanghai Acmec Biochemical Co., Ltd.), leaf alcohol (98% purity, Shanghai Acmec Biochemical Co., Ltd.), 1-nonanol (98% purity, Shanghai Acmec Biochemical Co., Ltd.), and cis-3-hexenyl acetate (98% purity, Shanghai Acmec Biochemical Co., Ltd.); four pheromones: (Z)-9-tetradecenyl acetate (93% purity, Shenzhen Regent Biochemical Technology Co., Ltd., Shenzhen, China), (Z)-11-hexadecenyl acetate (98% purity, Shanghai Bidd Medical Technology Co., Ltd., Shanghai, China), (Z)-7-dodecenyl acetate (90% purity, Shanghai Bidd Medical Technology Co., Ltd.), and (Z)-9-dodecenyl acetate (90% purity, Shanghai Bidd Medical Technology Co., Ltd.); and five pesticides: emamectin benzoate (90% purity, Energy Chemical), lufenuron (97.4% purity, Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China), chlorfenapyr (98% purity, Shanghai Yuanye Biotechnology Co., Ltd.), chlorantraniliprole (95% purity, Shanghai Yuanye Biotechnology Co., Ltd.), and lambda-cyhalothrin (96% purity, Guangdong Liwei Chemical Co., Ltd., Maoming, China). All chemicals used for testing and the fluorescent probe 1-NPN were dissolved in GC purify-grade methanol in a 400 µM solution. The 2.0 µM protein solution (in 10 mM HEPES and 100 mM NaCl buffer, pH 7.5) was titrated with 400 µM 1-NPN to a final concentration of 2–20 µM. The binding constant of the protein to 1-NPN was analyzed by assessing the continuous change of fluorescence value. Compounds of 2–20 µM concentration were added to the mixture of 2.0 µM 1-NPN and 2.0 µM protein or 2.0 µM 1-NPN alone to determine the binding rate of the ligands to the protein and the value of the fluorescence. The final fluorescence value was calculated as the fluorescence value after the addition of 1-NPN and protein with the competing ligand minus the fluorescence value after the addition of 1-NPN with the competing ligand only.

The dissociation constants (K_1-NPN) of SfruOBP31 were calculated using Graphpad Prism 8 software. The dissociation constant (K_i) of 1-NPN by competing ligands was calculated with the following formula using IC50 value: K_i = [IC50]/(1 + [1-NPN]/K_1-NPN). Here, IC50 is the concentration of competing ligands when the fluorescence intensity reaches half of the initial fluorescence intensity of 1-NPN, and [1-NPN] indicates the concentration of 1-NPN [16]. A threshold of K_i < 20 µM was recognized as binding.